Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 960

_____________________________ _____________________________

DNA Fragmentation in Cultured Cells Exposed to High Linear

Energy Transfer Radiation

BY

ERIK HÖGLUND

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2000

Dissertation for the Degree of Doctor of Philosophy, Faculty of Medicine, in BiomedicalRadiation Sciences presented at Uppsala University in 2000

ABSTRACT

Höglund, E. 2000. DNA fragmentation in cultured cells exposed to high linear energytransfer radiation. Acta Universitatis Upsaliensis. Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine 960. 58 pp. Uppsala. ISBN 91–554–4822–4.

The DNA double-strand break (DSB) is a critical lesion which, if not completely restored,can have serious biological consequences. The relative biological effectiveness (RBE) ofmany severe end-points are closely related to radiation quality, with increasedeffectiveness at elevated ionization density. Data presented provide information aboutthe influence of radiation quality on the initial processes causing DNA damage, and themechanisms leading to its restoration. Such information will increase the understandingof radiation action mechanisms in mammalian cells.

Human cells were irradiated with accelerated ions having linear energy transfer (LET)values in the range 40-225 keV/µm, and 60Co-photons. Detailed analyses of the DNAfragment distributions were performed in the size-range 5 kilobasepairs to 6megabasepairs by pulsed-field gel electrophoresis.

A non-random fragmentation of DNA was evident, with an elevated number of smalland medium-sized fragments for ion irradiation, and the total number of breaksincreased by 80–110% when these fragments were included in the analyses. The RBE forDSB induction was 1.2–1.5. A two-fold increase of the number of breaks induced pernitrogen ion passing the cell nuclues was found when LET was increased from 80 to 225keV/µm, indicating a possible role of particle track structure in DSB induction.Furthermore, the ability to repair DNA was closely related to radiation quality, with anincreased proportion of unrejoined breaks for densely ionizing radiation. Surprisingly,the majority of breaks were rapidly rejoined even following exposure to high-LETradiation. The proportion of breaks restored by the slow phase showed a five-foldincrease for the highest LET tested, compared with photons. The results presentednominates the complexity of breaks as one determining factor for reduced reparabilityreported following high-LET exposure.

Key words: DNA fragmentation, high linear energy transfer, DNA repair, double-strandbreak, complexity of breaks

Erik Höglund, Division of Biomedical Radiation Sciences, Department of Oncology, Radiologyand Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden

© Erik Höglund 2000

ISSN 0282–7476ISBN 91–554–4822–4

Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2000

To

Johanna and Alexander

This thesis is based on the following paper, which will be referred to in the text

by their roman numerals I–IV.

I. STENERLÖW, B., HÖGLUND, E. AND CARLSSON, J. Induction and rejoining of

large DNA fragments after ion irradiation. Radiation Research, 151, 642-648

(1999).

II. HÖGLUND, E., BLOMQUIST, E., CARLSSON, J. and STENERLÖW, B., DNA

damage induced by radiation of different linear energy transfer: initial

fragmentation. International Journal of Radiation Biology, 76, 539-547 (2000).

III. STENERLÖW, B., HÖGLUND, E., CARLSSON, J. and BLOMQUIST, E., Rejoining of

DNA fragments produced by radiations of different linear energy transfer.

International Journal of Radiation Biology, 76, 549-557 (2000).

IV. HÖGLUND, E. AND STENERLÖW, B., Induction and rejoining of DNA damage

after exposure to radiation of different linear energy transfer. Possible roles

of track structure and chromatin organisation. Radiation Research,

Conditionally accepted.

Reprints were made with kind permission from The Radiation Research Society

(I), and Taylor & Francis Ltd. (http://www.tandf.co.uk) (II and III).

CONTENTS

ABBREVIATIONS_____________________________________________ 7

INTRODUCTION _____________________________________________ 8

Tumour therapy by high-LET irradiation ____________________________ 9

Internal irradiation ________________________________________________ 9

External radiation ________________________________________________ 10

Radiation safety________________________________________________ 12

DNA damage and repair _________________________________________ 13

Cellular processing of the damage___________________________________ 14

AIMS OF THE STUDY ________________________________________ 16

METHODS __________________________________________________ 17

Cells _________________________________________________________ 17

Irradiation ____________________________________________________ 17

Low-LET photons ________________________________________________ 17

High-LET ions ___________________________________________________ 17

Quantification of induced DNA damage ____________________________ 20

Pulsed-field gel electrophoresis, PFGE_______________________________ 20

Conventional assay - the FAR assay ________________________________ 21

DNA fragmentation analysis_______________________________________ 22

RESULTS & DISCUSSION ____________________________________ 25

Induction _____________________________________________________ 25

FAR assay_______________________________________________________ 25

Fragment analysis ________________________________________________ 27

Rejoining _____________________________________________________ 35

Kinetics _________________________________________________________ 35

Rejoining after exposure to the same number of particles_______________ 40

Chromatin organization _________________________________________ 41

SUMMARY AND CONCLUSIONS ____________________________ 45

REFERENCES ________________________________________________ 47

ACKNOWLEDGEMENTS _____________________________________ 56

7

ABBREVIATIONS

bp base pair

δMi width of gel segment i

DNA deoxyribonucleic acid

DSB double-strand break

Fi fraction of DNA in gel segment i

Fslow fraction of breaks restored by the slow phase of repair

F<k fraction of DNA smaller than the threshold size k

FAR fraction of activity released

GCR galactic cosmic rays

HR homologous recombination

HZE high (H) atomic number (Z) and energy (E) particles

kbp kilobasepair

LET linear energy transfer

LMDS locally multiply damaged sites

Mbp megabasepair

Mi average fragment size in gel segment i

NHEJ non-homologous end-joining

OER oxygen enhancement ratio

PCC premature chromosome condensation

PFGE pulsed-field gel electrophoresis

RBE relative biological effectiveness

SEP solar energetic particles

τ½ DSB repair half-time

8

INTRODUCTION

Researchers have been fascinated by the nature of radiation, and its effects on

the environment and man, ever since the discovery of X-rays (Röntgen, 1895),

and of radioactive elements, polonium and radium, by Pierre and Marie Curie

in 1898. Although radiobiological research started in the context of medical

radiology, it gradually progressed to more fundamental questions concerning

the effects of different types of radiation on all types of biological systems.

Consequently, radiobiological research came to involve investigators from

various fields, not only biologists and clinicians, but also physicists and

chemists.

Already in the early 20th century, ionizing radiation was used in the hope of

eradicating tumours. The pioneers in the field explored its possibilities by a trial

and error approach, but as the available technology and scientific knowledge

improved, more sophisticated methods evolved. Developments — mainly in

physics and dosimetry — resulted in the concept of conformal radiotherapy, i.e.

the possibility of shaping radiation fields to accord with treatment volumes by

crossing several X-ray beams. It was soon realized that the physical properties

of charged particles, and their stopping in matter, could be used to improve the

therapeutic effects of radiation. At present, considerable efforts are being made

in the field of radiobiology, to elucidate the mechanisms governing how DNA

damage occurs and how it is repaired. Such knowledge could provide the

means to modify radiation response in both healthy and malignant tissues,

thereby improving the therapeutic effects still further.

It was also realized early on that ionizing radiation could also have adverse

health effects. An association between skin cancer and radiation among

radiologists were found as early as 1902, and much has been learned since then

about the risks of radiation, mainly from epidemiological studies on atomic

bomb survivors and radiation workers (see review by Ron (1998) and references

therein). Present-day radiation safety research is focused on risk assessment, e.g.

studies on the influence of low dose/long duration exposure to radiation of

different qualities.

Ionizing radiation can be grouped into two main categories: sparsely and

densely ionizing radiation. The qualitative property that distinguishes the two

9

is the spatial distribution of energy transfer in the surrounding matter. The

initial events, eventually leading to radiation induced biological effects, are the

ionization and excitation of atoms and molecules of the irradiated matter.

X-rays and gamma photons deposit their energy via secondary electrons, set in

motion by the incident photons at all depths in the target matter, and are

therefore sparsely ionizing. In contrast, the energy deposition of charged

particles such as α-particles and accelerated ions, is restricted to a limited

volume adjacent to the primary particle track, and a more densed ionization

pattern occurs. Linear energy transfer (LET) and related concepts have been

introduced to determine the energy deposited by charged particles in

microscopic regions. In 1962 the International Commission on Radiological

Units (ICRU) defined this quantity as follows:

"The linear energy transfer of charged particles in medium is the

quotient of dE/dl, where dE is the average energy locally imparted to the

medium by a charged particle of specific energy in traversing a distance of dl."

Tumour therapy by high-LET irradiation

Tumour therapy by high-LET radiation can be performed either internally, i.e.

endoradiotherapy — irradiation with nuclides located directly in the tumour

tissue — or by external beams of accelerated particles such as heavy ions, e.g.

carbon ions. Endoradiotherapy using radioactive nuclides holds promise for the

treatment of spread cancer disease, while external irradiation with high-LET

beams is more suitable for deap-seated, voluminous tumours.

Internal irradiation

The use of nuclides emitting high-LET particles in tumour therapy will

probably increase significantly in the future. Boron neutron capture therapy

(BNCT) has already been in use for a long time (reviewed by Carlsson et al.,

1992, and Barth, 1999). The rationale for BNCT is the likelihood that thermal

(slow) neutrons will be captured by the stable boron which is thereby activated.

A nuclear reaction that is initiated by the capture of neutrons, results in the

emission of short-range high-LET nuclear fragments — an α-particle and a

lithium ion — which deposit their energy within a few micrometres of the

10

decay (a distance comparable to the radius of a mammalian cell) with

considerable likelihood of cell killing.

Naturally radioactive nuclides can also be used for therapy. For instance,

α-particle emitters (e.g. 211At and 212Bi) have shown promising results in

pre-clinical studies, using a variety of labelled compounds (Vaidyanathan and

Zalutsky, 1996). Furthermore, nuclides emitting cascades of low-energy

electrons, viz. Auger emitters, can be useful for treatment (Daghighian et al.,

1996; Welt et al., 1996) by virtue of their proven ability to cause severe DNA

damage and thereby cell death, when situated within cell nuclei (Kassis et al.,

1987). The Auger cascade resembles in many ways the clustering of ionizations

that is characteristic of high-LET radiation.

Common to all of the internal radiation treatment modes outlined above, is the

need for effective targeting of the tumour cells by some kind of carrier

molecule. Of course, without strictly selective targetting at the malignant cells,

damage to healthy tissue will be unacceptable high. The prospects for cancer

therapy using radioactive nuclides were reviewed by Kampf (1990).

External radiation

One benefit of radiotherapy using external beams of light- and heavy ions, is

their proven high relative biological effectiveness (RBE) for reproductive cell death.

More important, the possibility of controlling the site of energy deposition of

charged particles, thereby inflicting radiation damage restricted to the tumour

area alone while sparing the surrounding healthy tissue, further improves the

therapeutic ratio (Tobias et al., 1982; Blakely et al., 1984). Furthermore, the effect

of oxygen on radiation sensitivity works in favour of tumour treatment using

radiation having high-LET, the explanation being that cells exposed to low-LET

radiation, such as X-rays, show a significantly increased radiation sensitivity

when molecular oxygen is present during the exposure. This effect is called

oxygen enhancement ratio (OER), defined as the ratio of hypoxic to aerated doses

needed to achieve the same biological effect. OER decreases with increasing

LET, from about 3–3.5 for cell killing with X-rays, to 1, i.e. no oxygen effect, for

α-particles at 150–200 keV/µm (Barendsen et al., 1966; Prise et al., 1990).

Irradiation with high-LET therefore overcomes the reduced sensitivity to

radiation damage expected of poorly oxygenated tumour cells.

11

External radiation for tumour treatment, using charged particles, started with

protons more than half a century ago, and proton therapy centres are now

operating all over the world. The first proposal to use protons for tumour

treatment was put forward as early as 1946 (Wilson, 1946), and pioneer

treatment started in the mid 1950s at the Lawrence Berkeley Laboratory

(California, USA) (Tobias et al., 1955). The historical development of proton

radiobiology and therapy was reviewed by Raju (1995), amongst others.

The use of charged particles in radiation therapy makes possible a better dose

distribution in tissue, compared with photons and electrons, due to their

inverse dose profile (i.e. the Bragg peak, see Figure 1). This increases the tumour

dose, while reducing the dose to the surrounding healthy tissue — a crucial

attribute, since the tolerance of the healthy tissue around the tumour is the

major limiting factor for tumour control. Furthermore, the narrow Bragg peak

can be modulated by means of absorbers, to achieve increased conformity with

the tumour tissue. This technique, with a modulated spread-out Bragg peak,

was first used in the late 1950s for treatment of human cancers by means of

protons (Larsson, 1962).

0

0.5

1

1.5

0 0.5 1 1.5 2

Abs

orbe

d do

se (a

rbitr

ary

units

)

Depth (mm)

Figure 1. Theoretical Bragg curve. Depth–dose distribution for a heavy charged

particle, stopped in water.

12

Most clinical experience with particles other than protons comes from

treatments at the BEVALAC accelerator in Berkeley (Calif., USA), using

helium, carbon and neon ions (Castro, 1995). Treatment centres where

accelerated ions are used (or are going to be used) have been set up at

numerous locations around the world, e.g. in Japan (Tsujii et al., 1997), in

Germany (Eickhoff et al., 1999), and Italy (Amaldi, 1998).

Regarding all treatment modalities, the dose to the tumour volume must be

sufficiently high for tumour control, while sparing healthy tissues in order to

prevent late side effects. For this purpose, beams of accelerated particles may be

an effective tool for external therapy of deep-seated tumours. The effect of the

treatment is governed mainly by its ability to produce irreparable damage to

the DNA. The LET dependence on induction and repair of DNA damage, in

both normal and malignant cells, following exposure to different particles

needs to be thoroughly explored in order to ascertain the most suitable

radiation quality to use. Too heavy a particle could produce irreparable damage

not only at the site of the tumour, but also in the channel of entry on its passage

through non-malignant tissue in front of the tumour, while lighter particles, e.g.

protons, may be insufficiently effective in the target volume. Extensive

investigations into the biological effects, in relation to radiation quality, may

help to optimize the outcome of tumour treatments using particle beams (Kraft,

1990; Blakely and Kronenberg, 1998; Kraft et al., 1999).

Radiation safety

Besides application to clinical treatment, high-LET radiation and its effects on

biological systems is highly relevant for radiation protection and safety. Several

epidemiological studies, performed mainly in mineworkers, have confirmed a

link between radon exposure (high-LET α-particles) and cancer in the lungs, as

reviewed by Jostes (1996). The relevance of such studies regarding the lower-

exposure home environment has been disputed and more information is

needed about the effect of low doses of high-LET radiation, regarding DNA

damage induction and repair. Complex damage, and subsequent mis-rejoining

or deletions, following high-LET radiation do cause mutations and

chromosomal rearrangements that in unfortunate cases lead to malignant

transformations. Knowledge about these mechanisms is vital for adequate

quantification of radiation risk.

13

Radiation safety is also discussed within the framework of space research.

Crews of manned space-craft are exposed to various kinds of ionizing radiation.

Two main sources of radiation in space are protons emitted as solar energetic

particles (SEP), and the galactic cosmic rays (GCR) consisting of protons and other

energetic nuclei — high (H) atomic number (Z) and energy (E) particles (HZE).

Since the physical aspects of the SEP protons and their interaction with matter

are fairly well established, measures can be taken to monitor their presence and

adequate estimation of the risk is possible. Thus, they do not constitute a

serious radiation protection problem, whereas the HZE particles present

significant problems for radiation protection. They have an energy of several

hundred MeV per nucleon, and high-LET nuclear fragments, produced on

impact with the shielding material, are highly penetrating and may severely

damage DNA in a large number of cells in the exposed crew. Further

information about the effects of HZE particles may make future risk estimation

more accurate and reliable, and help to define radiation limits for human

exploration of space (Schimmerling, 1992; Schimmerling, 1995).

DNA damage and repair

Ionizing radiation produces many different types of damage to macromolecular

species within cells, and the critical targets for radiation-induced cell killing are

located in the cell nucleus. This became evident in several experimental studies

where the cytoplasm and the nucleus were selectively irradiated. It is also

generally accepted that the critical target for generation of biological effects is

the DNA, where different types of lesions, including base damage, cross-links

(within the DNA molecule or with associated proteins) and strand breaks are

produced by the radiation. Of these lesions, the DNA double-strand break (DSB)

stands out as the critical one for radiobiological effects (reviewed by Iliakis,

1991). Furthermore, there is a close relation between radiation-induced DSBs,

chromosomal aberration production and cell transformation (Bryant, 1984;

Yang et al., 1989; Kiefer, 1992).

The consequences of an isolated lesion in DNA is not significant in terms of the

ultimate biological end-point, e.g. cell killing, because of effective cellular repair

mechanisms. However, a DSB is often associated with additional damage in the

immediate vicinity of the break, on either the same or the opposite strand,

14

forming more complex lesions caused by local clusters of ionizations. These

lesions have been called locally multiply damaged sites (LMDS) (Ward, 1981) and

their composition is closely related to radiation quality (Prise et al., 1994).

These qualitative differences in DSBs may provide some explanation for the

close relation between ionization density, i.e. LET, and a variety of biological

end-points. For instance, the RBE for reproductive cell death has been shown to

depend on LET, with RBE in the range 3.5–5 for LET values around 100–300

keV/µm (Wulf et al. 1985; Raju et al. 1991; Stenerlöw et al., 1995). Moreover, in

support of the proposed role of break complexity on the microscopic level, the

initial number of DSBs induced was found not to increase with LET (reviewed

by Prise et al., 1998). However, evidence of elevated levels of initially induced

DSBs has been reported by some investigators, using improved electrophoresis

protocols. These revealed a significant contribution from smaller DNA

fragments (200 kbp – 1 Mbp) that were not resolved in earlier studies (Löbrich

et al, 1996; Newman et al., 1997; Kraxenberger et al., 1998).

The smaller fragments have been suggested to derive from interactions between

the radiation, e.g. the track of a charged particle, and the higher order chromatin

structures of the genome. Even shorter fragments (<1 kbp), related to the basic

organization of DNA around the nucleosomes in the 30 nm chromatin fibre,

were predicted by theoretical analysis (Holley and Chatterjee, 1996). Such

fragments have also been detected experimentally by Rydberg et al. (1996). An

extensive exploration of DNA fragmentation, in relation to radiation quality

and genomic organization, may facilitate adequate modelling of the

development of DNA damage. This could help extrapolation to effects from low

doses, or even single events, and such information would be valuable for risk

estimation in radiation safety (Friedland et al., 1998; Sachs et al., 1998).

Cellular processing of the damage

The repair of DNA damage is a critical step that could lead from the initial

damage, to broken or rearranged chromosomes, cell death, or cancer. In order

to prevent this, the cells have evolved various ways to restore the genome, and

early evidence of the occurrence of repair of X-ray induced damage in

mammalian cells was provided by Elkind and Sutton (1960).

15

It is well known that high-LET irradiation can cause damage that is more

difficult to repair, and that this largely explains the high RBE values for severe

outcome. The explanation of the restricted repair capacity is believed to be a

greater complexity of induced DSBs, due to clustered damage (Roots et al., 1979;

Ward, 1985; Goodhead, 1989; Goodhead, 1994; Ward, 1994). Indeed, slower

repair and a larger proportion of residual damage following high-LET exposure

have frequently been reported (Ahnström and Edvardsson, 1974; Ritter et al.,

1977; Coquerelle et al., 1987; Blöcher, 1988; Heilmann et al., 1993; Jenner et al.,

1993; Weber and Flentje, 1993; Stenerlöw et al., 1996), and non-repairable DSBs

and unrejoined chromatin fragments are closely correlated to cell killing.

The increased complexity may lead to misrejoining of breaks, i.e. two ‘wrong’

ends are joined together by the cellular repair mechanism. Several end-points

that are believed to require misrejoining events, e.g. chromosome exchanges

(Durante et al., 1992; Durante et al., 1995) and interstitial deletions (Thacker,

1992), are induced at increased frequencies by increasing LET values. A possible

explanation may be found in the clustering of breaks seen for high-LET. The

numerous breaks within a cluster, or in the immediate vicinity of a high-LET

particle track, are more likely to be rejoined with a ‘wrong’ end, suggesting the

involvement of higher order chromatin structures in the formation of

chromosome aberrations derived from induced DSBs (Sachs et al., 1997).

16

AIMS OF THE STUDY

The DNA double-strand break is a critical lesion which, if not completely

restored, can have serious biological consequences, including cell death,

chromosomal aberrations, mutations, and cellular transformation. Moreover,

the relative biological effectiveness (RBE) of these end-points has been shown to

be closely related to radiation quality, with increased effectiveness at elevated

ionization density. Better knowledge of the initial processes causing DNA

damage, and the mechanisms leading to its restoration, should help us to

identify critical lesions and increase our understanding of radiation action

mechanisms in mammalian cells. The principal aim of this work was to

investigate the influence of radiation quality on the induction and processing of

DNA damage in mammalian cells in vitro, taking DNA fragment distribution

into account. In greater detail:

• to investigate the initial fragmentation of DNA following exposure to the

same type of ions having different LET values;

• to investigate the rejoining of DNA fragments following exposure to the

same type of ions having different LET values;

• to investigate the induction and rejoining of DNA double-strand breaks

following exposure to different ions having similar LET values;

• to investigate the possible involvement of particle track structure and

higher order chromatin organization in the induction and rejoining of DNA

double–strand breaks.

17

METHODS

Cells

Low-passage normal human fibroblast cells (GM5758, Human Genetic Mutant

Cell Repository, Camden, NJ, USA) were used as a model system in most of the

presented work (Papers II–IV). The cells, which were labelled with 14C or 3H

and grown to confluence prior to irradiation, constituted a homogeneous

system, suitable for investigation of DNA damage. Furthermore, since they are

not very specialized, they share many characteristics with other kinds of cells in

the body. In the first paper (Paper I) two human cell lines were used, U-343MG

glioma and K562 erythroleukemia cells.

Cells were irradiated as monolayer cultures (DNA DSB rejoining and

induction), or embedded in agarose plugs (DNA DSB induction). All irradiation

was performed on ice to prevent any repair processes during exposure.

Irradiation

Low-LET photons

Throughout this work we have used photons from a conventional 60Co

apparatus (1.2 MeV photons, LET ~0.5 keV/µm) as reference radiation. The

photon dose rate was 1–1.3 Gy per minute, as determined by ionization

chamber and thermoluminiscence dosimetry.

High-LET ions

The Gustaf Werner synchrocyclotron at the The Svedberg Laboratory (Uppsala,

Sweden) provided the accelerated ions. The different ions (14N6+, 14N7+, 10B5+ and20Ne9+) were extracted from external ion sources and accelerated to energies of

about 32–48 MeV/u. Beam properties are listed in Table 1. The ion beams were

directed, from the accelerator to the Biomedical Unit, through a high vacuum

beam transport system.

18

ED C B A

beam direction

Figure 2. Ion-beam delivery. Experimental set-up for ion irradiation at the

Biomedical Unit, The Svedberg Laboratory, Uppsala University (see Stenerlöw

et al., 1996 for details). Gold foil (A), transmission chamber (B), plastic

absorber (C), scanning device for Markus chamber (D) and ice-cooled sample

holder (E). [By courtesy of T. Hartman, The Svedberg Laboratory, Uppsala,

Sweden.]

On entering the experimental area the beams were quite narrow (~ 1 cm in

diameter) and had to be scattered laterally by a thin gold foil (A, Figure 2) to

achieve a uniform dose distribution at the position of the sample (E). The beam

profile was analysed by vertically and horizontally scanning an ionization

chamber (D) over the target area, and the dose distribution in the target was

uniform ±5% within a radius of 18 mm. Absolute dosimetry was performed

using a transmission chamber (B) together with the ionization chamber (D) (see

Stenerlöw et al. (1996) for details.)

When heavy charged particles traverse matter, they lose energy mainly by

electromagnetic interaction with the electrons in the target material. The rate of

energy loss for the particle is proportional to the square of its charge (Z2) and

inversely proportional to the square of its velocity (β2). Thus, the magnitude of

the dose absorbed by the medium increases with decreasing particle speed, and

the resulting depth–dose distribution has the shape of the classical Bragg curve

(Figure 1). The depth–dose distribution and the maximum range of the particles

was determined by placing plastic absorbers (C) of varying thickness in front of

the ionization chamber and plotting the registered dose vs the thickness of the

absorber.

19

Cells were irradiated at different positions on the Bragg curve by placing

absorbers of differing thickness in front of the samples. The corresponding ion

energies and LET values (Table 1) were obtained from measurements of the

residual beam range and calculations of the stopping of ions in water (Ziegler

and Manoyan, 1988).

Table 1. Beam properties

Radiation

Ion energy at cells

(MeV/u)LETa at cells

(keV/µm) Paper

60Co photons - < 0.5 I-IV

Helium ions 0-22 40 (35-55)b II

Boron ions 37

16

40

80

unpublished*

Nitrogen ions 36

16-25

14

10

80

125 (110-150)c

175

225

II-IV

I-IV

II-IV

II-IV

Neon ions 24

13

225

300unpublished*

a Unrestricted.b Extended Bragg peak. The LET range stated covers a 68% confidence interval.c Cells in agarose plug (1 mm thick).

* Manuscript in preparation, Höglund, E. and Stenerlöw, B., 2000.

20

Quantification of induced DNA damage

Pulsed-field gel electrophoresis, PFGE

PFGE has found a broad application in biochemistry and genetics. One of its

greatest advantages has been its sensitivity in the analysis of long DNA

molecules. Fragments up to 13 Mbp long have been separated (Orbach et al.,

1988), and the ability to separate such large fragments makes it possible to

study radiation-induced DNA fragmentation, and cellular processing of

damage, following exposure to doses similar to those used in cell survival

experiments. For instance, rejoining of radiation-induced DSBs has been

measured in cultured cells after irradiation with doses as low as 5 Gy

(Stenerlöw et al., 1996).

In order to separate megabasepair-length DNA fragments by size, pulsed

electric fields with periodically changing direction were applied. The optimal

angle between the two field directions has been shown to be near 120°

(Schwartz and Cantor, 1984; Southern et al., 1987; Cantor et al., 1988) which was

the angle used in the present work. The exact mechanisms involved in the

separation have not yet been extensively described, but some intuitive

reasonings might be illustrative. Since the fragments are much longer than the

average size of the pores in the agarose matrix, the retarding effect is of minor

importance in a static field, i.e. two fragments of different length will migrate

with the same velocity. But, when the pulsed fields were applied, the fragments

aligned alternately with the two field directions, always with their trailing ends

first because of the lower force of friction experienced by the fragment ends at

the obtuse-angled corner. Thereby shorter fragments started from a more

advanced position and gained ground in every pulse, thus resulting in a size

separation of the fragments.

By changing various parameters, e.g. pulse duration, field strength or agarose

concentration, the PFGE protocol can be optimized for separation of differently

sized fragments. Longer pulse times and weaker electric fields favour longer

fragments, while an increased agarose concentration favours shorter ones.

Three different protocols were used in this work. First, the very long fragments

(Paper I) were separated using a 74-hour run at 1.4 Volts per cm. In the more

detailed analyses of the fragmentation, performed in the succeding

investigations (Papers II-IV), we needed distinct separation within a wide

21

sizerange. To achieve this we chose two shorter schemes, 45.7 and 17 hours (see

Paper II and Table 2 for details) which, when combined, provided separation of

fragments in the range 5 kbp up to about 6 Mbp with good resolution.

Table 2. Pulsed-field gel electrophoresis protocols

Optimal

size range

Field

(V/cm)

Pulses

(length/duration)

Total time

(h) Paper

< 10.5 Mbp 1.4 180-60 mina 74 I

1 - 6 Mbp 2 10 min/ 3 h

20 " / 5.33 h

30 " / 8 h

40 " / 9.33 h

60 " / 20 h

45.67 II-IV

5 kbp – 1.5 Mbp 7 10 s / 7 h

40 " / 5 h

70 " / 5 h

17 II-IV

a Pulses were ramped, i.e. they were continuously shortened from 180 to 60 minutes during the 74 h

electrophoresis run

Conventional assay — the FAR assay

Various techniques have been used to measure the yields of DNA DSBs

following exposure to radiation (reviewed by Prise et al., 1998). One of the most

widely used methods is the conventional FAR assay, in which the Fraction of

Activity Released from the sample plug is used as a measure of the degree of

damage. The FAR value designates the amount of radioactively labelled DNA

that enters the gel during the electrophoresis run. Only DNA molecules smaller

than a certain exclusion size can enter the gel, leaving very large fragments and

undamaged DNA in the plug. Specific DNA size markers, e.g. the largest

chromosome of Schizosaccharomyces pombe (5.75 Mbp), were also used to

determine the FAR value. The PFGE protocols used are optimized for

separation of Mbp-sized fragments, and the size distribution of the migrated

fragments is disregarded. The number of induced DSBs is related to the FAR

22

value in a non-linear fashion, and an assumption of randomly distributed

breaks along the DNA molecule gives the breakage yield, expressed as breaks

per unit length of DNA and dose (Blöcher, 1990; Cook and Mortimer, 1991;

Cedervall et al., 1995). The random breakage model used throughout this work

is Blöcher’s model (1990) :

))(( nk

nrk

k 11e1F nrk

−−−−++++−−−−==== −−−−<<<< , (1)

where kF<<<< is the fraction of DNA whose size is smaller than threshold size k, r

is the mean number of DSBs per chromosome, and n is the average size of a

chromosome.

DNA fragmentation analysis

Two major drawbacks of the FAR assay are the assumption of random

fragmentation, and the poor resolution for separation of smaller DNA

fragments. These are problems of great importance when analysing DNA

damage after exposure to high-LET radiation, due to an increased likelihood

that short and medium-sized DNA fragments will be produced, compared with

predictions based on random breakage models (Prise et al., 1998). The shorter

fragments are a direct result of the interaction between the particle track

structure and all levels of chromatin organization in the cell (Rydberg, 1996).

The conventional assays, which do not include these smaller fragments, thereby

tend to underestimate the RBE for DSB induction. This is also evident in the

literature (Löbrich et al., 1996; Newman et al., 1997). The RBE is defined by the

quotient of the yields for the test radiation and the 60Co reference radiation.

Thus, a careful investigation of the DNA fragment size distribution is necessary

for correct scoring of the damage.

23

S. cerevisiae

λ DNA-PFGE

Migrated DNA

48 97 145 225 375 680 9301100

Size

Mig

rate

d D

NA

Figure 3. Gel slicing. The lanes of migrated DNA were cut into different size

segments, according to DNA size markers loaded in the gels. Sizes are listed in

Table 3. The figure illustrates the gel optimized for separation in the range 5

kbp – 1.5 Mbp, and the markers S. cerevisiae and Lambda DNA-PFGE were

used.

The lanes of migrated DNA were cut into ten different size segments according

to DNA size markers (S. pombe, S. cerevisiae and Lambda DNA-PFGE markers)

loaded in the gels (listed in Table 3). In Figure 3, the slicing of the gel optimized

in the range 5 kbp–1.5 Mbp is illustrated. The number of DNA fragments (n) in

each size zone is proportional to the mass of DNA in the corresponding gel

segment. By dividing the fraction of DNA in a gel segment (Fi) by the average

fragment size in that zone (Mi), the number of fragments in each size zone can

be calculated according to Eq. 2 (see Paper II for details).

(((( ))))i

ii

M

FMn ≅≅≅≅ (2)

24

Table 3. Gel segments

DNA size intervaliM

(kbp)iMδδδδ

(kbp)

<48 24 48

48-97 73 48

97-145 121 48

145-225 185 80

225-375 300 150

375-680 527 305

680-930 805 250

930-1110 1020 180

1110-3500a 2305 2390

3500-5750 4625 2250a This segment originates from both PFGE gels.

The fraction of DNA <1110 kbp in the 5–1500 kbp gel was subtracted from the fraction of DNA<3500 kbp in the 1–6 Mbp gel.

The total number of DNA DSBs induced for fragments up to 6 Mbp was

obtained by cumulatively adding the number of fragments for each individual

size interval. This was done for all doses and radiation qualities and, from the

dose–response curves, the DSB yields could be calculated by linear regression.

25

RESULTS & DISCUSSION

Induction

FAR assay

The dose response for the the FAR value, i.e. the amount of DNA that migrates

into the electrophoresis gel, depends on several conditions. Besides the

electrophoretic parameters, such as agarose concentration and field strength,

the threshold size chosen for the analysis can also affect the outcome (Paper I).

Further, the mode of DSB induction determines the dose response. If the breaks

are distributed randomly along the chromosome, an induction of DSBs linear

with dose will result in a non-linear fraction of extractable DNA (Blöcher, 1990).

0

0.05

0.1

0.15

0.2

0 5 10 15 20

nitrogen, U-343MGγ rays, U-343MGnitrogen, K562γ rays, K562

Frac

tion

of D

NA

rele

ased

<6

Mbp

Dose (Gy)

Figure 4. Dose response. The fraction of DNA released for the molecular weights <6

Mbp, is shown for K562 and U-343MG cells irradiated with nitrogen ions

(125 keV/µm) and 60Co-photons. Data for both cell lines were fitted to one

single random-breakage curve after photon irradiation. Following nitrogen ion

exposure, separate linear curves were fitted for each cell line.

(Data from Paper I.)

26

The dose response, as determined by the fraction of DNA smaller than 6 Mbp,

was compared for two human cell lines (U-343MG and K562) after exposure to

low-LET gamma and high-LET nitrogen ions (Figure 4). The data representing

the ion irradiation showed a linear dose response for doses below 30 Gy, in

contrast to the sigmoid-shaped random-breakage curves seen for gamma. The

ion data could not be satisfactorily described by a model assuming random

breakage and were best fitted to linear expressions. Instead, the γ and ion

curves intersected at 12 and 18 Gy for the K562 and the U-343MG cells,

respectively. This phenomenon has not been noted earlier by investigators

using either pulsed or constant-field gel electrophoresis, probably because the

fraction of DNA migrating out of the plug — without any specific DNA size

marker — was used as a measure of the damage, resulting in a relatively large

threshold in their analysis (e.g. Rydberg et al., 1994). In some studies where the

neutral filter elution technique was used, similar linear dose responses were

reported for 238Pu α-particles (Prise et al., 1987) and nitrogen ions (Nygren and

Ahnström, 1996). The reason why the intersections were found at different

doses for the two cell lines in Paper I is unclear. One possible explanation might

be differences in higher-order chromatin structures and organization between

the cell lines. Nevertheless, it is evident from the present work that

nonrandomness of DSB induction by high-LET radiation can also be detected

by the conventional FAR assay.

Low random yield and clustered breaks

The relative yields for fragment sizes smaller than 6 Mbp, calculated by

dividing the ion dose–response curves by the gamma ditto, significantly

exceeded unity (~ 3 at 1.5 Gy) for doses up to about 10 Gy, but decreased at

higher doses (~ 0.8 at 30 Gy). For sizes 6–10.5 Mbp the correlation was reversed,

with a constant ratio around 0.7, i.e. the ions were less effective at producing

large fragments. A possible interpretation of the results could be that the

induction of DSBs following high-LET irradiation includes not only a random

breakage mode, but also a non-random component. It can be assumed that, for

each randomly induced break, one (or more) additional associated break formes

within a certain distance from the original break. If this distance is smaller than

the chosen threshold, these fragments can enter the electrophoresis gel and

thereby contribute a linear dose response to the fraction of DNA released. Based

on these simple assumptions, the data for the nitrogen ions were modelled. It

was found that when a relatively low random induction yield was used (about

50% of the yield measured for 60Co-photons) together with s associated breaks

27

within x Mbp from the initial random break, the nitrogen data could be fitted

for doses up to 30 Gy when sx = 2 Mbp (Paper I). The product sx could be

interpreted as a release of either one single fragment of size 2 Mbp, or several

smaller fragments of total length 2 Mbp. Although no extensive conclusions

should be drawn from such a rough analysis, the result supports the idea of

clustered DSBs within higher orders of chromatin structure, as proposed by

many others (Jin et al., 1995; Löbrich et al., 1996; Newman et al., 1997;

Kraxenberger et al., 1998; Frankenberg et al., 1999). The relative influence of the

non-random component will increase for decreasing doses, because at lower

doses the random breakage produces fragments that are too large to be released

below the chosen threshold. This may also explain why the linear response for

high-LET at low doses has not been recognized earlier — the analyses have

usually been performed using higher doses (e.g. Heilmann et al., 1995). In order

to further analyse the significance of these small and medium-sized fragments

for the DSB induction yield, it is essential to take the size distribution of

fragments into account.

The significant non-random contribution to the induction yields for high-LET

was also confirmed in Paper II, in which a more detailed investigation of the

LET dependence of DNA fragmentation in normal human fibroblast cells (GM

5758) was performed. For this cell line too, a linear dose response was found

when DNA sizes below 1.11 Mbp were measured with the FAR assay for

particle irradiation up to 50 Gy, while the 60Co data were consistent with a

random breakage model.

Fragment analysis

To further examine the dependence on radiation quality, cells were exposed to

accelerated nitrogen ions at four different LET values in the range 80–225

keV/µm. Helium ions having an average LET of 40 keV/µm were also used,

but their results will not be discussed further in this presentation, as the helium

beam consists of a mixture of low and high-LET particles.

Non-random fragmentation

The frequency of DNA fragments over the molecular weight range investigated

was calculated for several doses in the range 0–200 Gy for all radiation qualities

tested. Examples of the results for 125 keV/µm nitrogen ions at 50, 70 and 100

Gy are plotted in Figure 5a. Here the data calculated for each size interval (Eq.

28

2) were divided by δMi (see Table 3) and plotted against fragment size, so that

the area under the curves corresponds to the number of fragments induced per

basepair. The high-LET fragmentation patterns did not show any resemblance

to the distributions expected when the breaks had been induced randomly

along the DNA molecules (dotted curves in Figure 5a). Further, the mode of

DSB induction was examined for the two radiation qualities by calculating the

random DSB yield for measured levels of the fraction of DNA smaller than

different threshold sizes (Figure 5b). The resulting yields for the different sizes

could intuitively be perceived as follows; to produce the measured amount of

DNA smaller than these sizes by random breakage alone, the calculated yields

would be needed. Thus, if the breaks were induced randomly over the genome,

DSB yields would be independent of the fragment sizes studied and the result

would be a horizontal line in Figure 5b. With small deviations, the result for

photons followed a similar curve, with a DSB yield around 6 × 10-9 DSBs bp-1

Gy-1, regardless of threshold size (cf. Table 4), while the high-LET curve

revealed an increased likelihood that shorter DNA fragments would result. For

instance, an RBE of about 2.5 for DSB induction was measured when analysing

fragments smaller than ~100 kbp, and the RBE decreased to below unity when

increasing the threshold to sizes greater than 2–3 Mbp.

29

0.01

0.1

1

10 100 1000 104

125 keV/µm (100 Gy)125 keV/µm (70 Gy)125 keV/µm (50 Gy)

Freq

uenc

y of

DNA

frag

men

ts (x

10-1

2 bp-2

)

Fragment size (kbp)

a

0

5

10

15

20

100 1000

125 keV/µm N-ionsCo-60 photonsDN

A d

sb y

ield

( x1

0-9 d

sb b

p-1 G

y-1)

Threshold size (kbp)

b

Figure 5. Excess of short and medium-sized fragments. (a) Frequency of DNA

fragments as a function of mean fragment size, following exposure to different

doses of 125 keV/µm nitrogen ions. Dotted lines represent the corresponding

fragment distributions, as expected from random breakage (Eq. 1) with a DSB

yield of 5.8 × 10-9 DSBs bp-1 Gy-1. (b) Induction yields calculated with the

random-breakage formula (Eq. 1), using varying threshold sizes (see main text

for details). (Data from Paper II.)

30

Due to the evident excess of short and medium-sized fragments following

exposure to densely ionizing radiation, the total number of breaks has to be

integrated over all fragment sizes, i.e. by fragment analysis. The risk of

underestimating the number of induced DNA DSBs could thereby be

diminished. The results are listed in Table 4 (data from Papers II and IV), and

Table 5 (data from Höglund and Stenerlöw*)

Table 4. DNA double-strand break induction yields after irradiation at different

linear energy transfer (LET)

Radiation LET(keV/µm)

Yield±SEa

(10-9 DSB/bp/Gy)

Yield±SEb

(DSB/traversal)

RBEc

DSB induction60Co < 0.5 5.8±0.1 - 1

N ions 80 8.6±0.9 4.5±0.2 1.5

125 8.6±0.3 6.7±0.2 1.5

175 7.6±1.0 8.4±0.9 1.3

225 6.9±0.7 10.0±0.3 1.2a Calculated using DNA fragmentation analysis.

b The yield as expressed by the number of breaks induced per particle traversal per cell, assuming a

cross-sectional area of the cell nucleus of 150 µm2 and 6x109 bp per diploid cell in G0/G1.c RBE for DSB induction was defined as the quotient between the yield for the test irradiation and

the 60Co-reference yield.

31

Table 5. DNA double-strand break induction yields after irradiation with

different ions having different linear energy transfer (LET)

Radiation LET(keV/µm)

Yield±SEa

(10-9 DSB/bp/Gy)

Yield±SEb

(DSB/traversal)

RBEc

DSB induction

B ions 40 9.2±0.4 2.4±0.1 1.6

80 8.3±0.2 6.7±0.1 1.4

Ne ions 225 5.4±0.2 7.8±0.2 0.9

300 6.6±0.3 12.8±0.5 1.1a Calculated using DNA fragmentation analysis.

b The yield as expressed by the number of breaks induced per particle traversal per cell, assuming a

cross-sectional area of the cell nucleus of 150 µm2 and 6x109 bp per diploid cell in G0/G1.c RBE for DSB induction was defined as the quotient between the yield for the test irradiation and

the 60Co-reference yield.

The DSB induction yields, scored by fragment analysis, showed a two-fold rise

(80–110%) for the ions, compared with use of conventional methods based on

random breakage (e.g. the FAR assay), while the corresponding increase for60Co-photons was only 13%. Consequently, RBE attained values significantly

above unity for the ions at all LETs, even though they decreased for the highest

LETs tested (Table 4 and Figure 6). On the other hand, the RBE values assessed

by conventional assay did not exceed unity for any of the LETs tested. The RBEs

were defined as the quotients of the DSB induction yields for the test radiation

qualities versus the 60Co reference radiation. The increase in RBE values for DSB

induction by high-LET particles presented in this work cannot fully explain the

sometimes very high RBEs reported for a variety of biological end-points,

including cell killing, mutation induction (Cox et al., 1977; Tobias et al., 1982;

Blakely et al., 1984; Kiefer et al., 1992; Lett, 1992) and cell transformation (Yang et

al., 1997). It is generally believed that a decreased ability to maintain the

integrity of the genome by the repair mechanism, due to an increased

complexity of breaks, can lead to misrejoining events and loss of genetic

material to a greater extent for high-LET radiation (Goodhead et al., 1993). This

still seems be to be the main cause of the higher RBE values scored several cell

generations later.

32

0

0.5

1

1.5

2

0.1 1 10 100

RB

E, d

sb in

duct

ion

LET (keV/µm)

Figure 6. RBE for DSB induction. The RBE as a function of ionization density for60Co-photons and 80-225 keV/µm nitrogen ions, calculated by fragment

analysis (�). The corresponding RBE values calculated without taking the

fragmentation into account, based on the fraction of DNA <6 Mbp and

assuming random distribution of breaks, are also included (�). The lines

connecting the points are plotted for guidance only, and do not assume any

particular behaviour of the RBE/LET relation. RBE determined by fragment

analysis and published by others is included for comparison. Nitrogen ions at

97 keV/µm (�) and iron ions at 150 keV/µm (�) (Löbrich et al, 1996), and

110 keV/µm α-particles (�)(Newman et al. 1997).

The influence of single-hit events, or intra-track effect, is more pronounced for

high-LET tracks than for the more dispersed ionization produced by photons.

Consequently, additional fragments are produced in the same DNA molecule

by the same particle traversal. Thus, for the same dose, the ions prove to be less

efficient for production of fragments of greater molecular weight (>1 Mbp),

compared with photons, or than expected from random breakage alone (e.g.

Figure 5b). The explanation is probably to be found in the nature of the particle

tracks, with different track structures at different LET, and their interaction

with the genomic structure on several levels in the cell. The importance of DNA

structure inside the 30-nm chromatin fibre for the production of very small

33

fragments (80 bp – 2 kbp) has been shown in both theoretical (Holley and

Chatterjee, 1996) and experimental work (Rydberg, 1996). Such small fragments

could not be detected by the methods used in the present work, due to a lower

limit of about 5 kbp for fragments not migrating out of the electrophoresis gels,

but the contribution to the total yield of DSBs from these small fragments is

limited (see discussion in Paper III and Rydberg, 1996). However, the

higher-order chromatin structure may not be the chief cause of the release of

small and medium-sized fragments. Theoretical presentations, assuming a

random-coil structure of the DNA molecule, have also predicted a significant

increase in correlated breaks, i.e. breaks induced within the same molecule by

one particle track, compared with more sparsely ionizing radiation

(Hutchinson, 1996; Sachs et al., 1995). Experimental data on fragmentation of

naked DNA may provide important information on the role of higher-order

chromatin structure.

Efficiency for DSB induction per particle

Furthermore, when delivering the same dose to the cells for different LET

values, the numbers of traversing ions differ, e.g. a dose of 1 Gy to the cell

nucleus is given by approximately 12 ions at 80 keV/µm, while the same dose is

received when 5 ions traverse the nucleus at 175 keV/µm. To account for this,

and to further clarify LET dependence on DSB induction, the cells were exposed

to nitrogen ions at four different LET values (80–225 keV/µm, Paper IV), and

with boron ions (40 and 80 keV/µm) and neon ions (225 and 300 keV/µm) (data

from Höglund and Stenerlöw*). The effectiveness to cause DNA damage was

analysed for each particle traversal. In contrast to yields expressed as a function

of dose (Paper II), the effectiveness to induce DSBs per particle traversal per cell

increased with increasing LET for the same type of ion (Table 4, 5 and Figure 7).

Further, the heavier neon ions were less effective DSB inducers, compared with

nitrogen ions at the same LET (225 keV/µm, Figure 7). An immediate

conclusion from this is that the slower ions having higher LET values (or the

lighter ions having the same LET value) are more effective DSB inducers due to

the more dense ionization pattern in their tracks. This is in accordance with

earlier studies where low energy protons were more effective mutation

inducers than were α-particles (Belli et al., 1992) or neon ions (Durante et al.,

1998) at the same LET. Also singly charged protons and deutrons, matched in

LET, have proved to be more effective for cell inactivation than doubly charged

particles of the same LET (Folkard et al., 1996). However, at 80 keV/µm, no

significant difference was detected between irradiation with boron or nitrogen

34

ions. This type of analysis and presentation provides information about DNA

fragmentation at the low-dose limit of a single particle traversal per cell.

0

2

4

6

8

10

12

14

50 100 150 200 250 300

Boron ionsNitrogen ionsNeon ions

Yiel

d (D

SB/p

artic

le/c

ell)

LET (keV/µµµµm)

Figure 7. DSB induction per ion traversal, in this instance for boron, nitrogen and

neon ions having different LET values. (Data from Paper IV. Boron and neon

data from Höglund and Stenerlöw*).

35

Rejoining

Kinetics

As described in the previous section, the significant increase in DSB induction

yield for high-LET, reported in this work and elsewhere, cannot fully explain

the relatively high RBE values reported for many other end-points. In the

analysis of DNA repair, such additional breaks might have influenced the

rejoining kinetics. It could be argued that the increased number of DNA

fragments, originating from regional clusters of breaks following high-LET

exposure, will impede the cellular repair mechanism in effectively rectifying the

damage due to enzymatic saturation, but only by virtue of a too large number

of breaks to be repaired. Furthermore, a greater complexity of breaks (Ward,

1985; Goodhead, 1989; Goodhead, 1994; Ward, 1994) may restrict repair

capacity (Roots et al., 1979), with consequent misrepair events, which in turn

would promote, e.g. cell death and mutation induction. Many studies on DSB

repair have shown a slower rejoining of fragments, and a larger proportion of

residual breaks following high-LET irradiation (Ritter et al., 1977; Coquerelle et

al., 1987; Blöcher, 1988; Heilmann et al., 1993; Jenner et al., 1993; Weber and

Flentje, 1993; Stenerlöw et al., 1996). However, in order to detect a possible effect

on DSB rejoining from smaller DNA fragments and/or break complexity in

relation to LET, the fragment size distributions have to be included also in

investigations on rejoining.

The way in which DSB rejoining kinetics and the level of unrejoined DSBs was

dependent on LET in human fibroblast cells, following 0–20 hours of

post-irradiation incubation was investigated by fragment analysis (Paper III).

The fragment analysis has been described above. Briefly, the fragment size

distributions were taken into account by summing up the contributions of all

size intervals, and the total number of DSBs for each time point was calculated.

Rapid rejoining following high-LET

As expected from earlier investigations, the rejoining of DSBs showed a

biphasic behaviour for all radiation qualities tested, but surprisingly, and in

contrast to previous investigations, a pronounced rapid rejoining component

which accounted for most of the rejoining could be detected also following

exposure to densely ionizing radiation (Figure 8). Half-times were similar (~15

minutes) for all radiation qualities tested. More consistent with previous

36

studies, both the proportion of unrejoined DSBs and the fraction of breaks

rejoined by the slow phase (Fslow) increased with LET, and the half-time for the

slow kinetics was 2–3h. Some of the rejoining parameters from Paper III are

summarized in Table 6.

Table 6. DSB rejoining parameters following irradiation with 100 Gy of

different radiation qualitiesa

Radiation LET

(keV/µm)

τ1/2b (fast)

(min)

Fslowc Unrejoined (%)d

60Co <0.5 19 ± 8 0.07 ± 0.03 4 ± 3

N ions 80 17 ± 2 0.16 ± 0.04 5 ± 2

125 13 ± 2 0.36 ± 0.10 7 ± 4

175 15 ± 7 0.29 ± 0.02 12 ± 4

225 12 ± 4 0.38 ± 0.05 12 ± 2a Based on fragment analysis for sizes up to 5.7 Mbp (±SE). The parameters were obtained from

double-exponentional curves fitted to the data.b Time to halve DSB level for the rapid phase of rejoining. The number of unrejoined DSBs at 20–22

hours was subtracted to measure the kinetics of rejoinable breaks only.c Fraction of DSBs rejoined by the slow phase.

d Proportion of initially induced DSBs, measured by fragmentation analysis.

Although Fslow increased significantly with LET vis-à-vis photon irradiation,

showing a five-fold increase at 225 keV/µm, the fractions measured were

smaller than earlier reported results (e.g. Stenerlöw et al., 1996). The main cause

of the difference is that the smaller fragments (<1 Mbp), induced by spatially

correlated DSBs, were revealed and included in the present work by fragment

analysis. This yielded a significant increase in the degree of initial damage

(Paper II) that was not detected by earlier conventional assays. However, within

a few hours of repair, both methods showed similar levels of damage (Figure 8),

and it was clear that the majority of the breaks were rejoined in the rapid phase

even for the high-LET ions. This may also explain the rather low levels of

unrejoined breaks presented (Table 6).

37

0

200

400

600

800

1000

0 1 2 3 4 5 6 7

125 keV/µµµµm nitrogen ionsBlöcher repair

Time (h)

Num

ber o

f DSB

(x10

9 bp-1

)

22

Figure 8. Rapid rejoining for high-LET. The number of DSB per Gbp (109 bp) as a

function of post-irradiation incubation time. Cells were irradiated with 125

keVµm nitrogen ions and the number of breaks was determined using

fragmentation analysis (see main text for details). The dotted curve is included

for comparison, representing the number of breaks as determined by Blöcher’s

conventional formula (Eq. 1).

The rapid rejoining described that was evident after high-LET exposure should

not be interpreted as an indication of a quicker rejoining of short DNA

fragments, in comparison with the rejoining of longer fragments. When large

numbers of short fragments are joined to other shorter fragments, or to larger

pieces of DNA, longer fragments are formed. In this way they gradually move

through the fragment size distribution into gel segments representing

increasing fragment sizes, with the net effect of an apparent rapid removal of

shorter fragments. Furthermore, in fragment analysis, the number of fragments

smaller than 6 Mbp is used to estimate the number of DSBs. This is a high-dose

approximation, and at low doses and/or long repair times, there is a risk of

underestimating the number of DSBs. The reason is that the longer fragments,

produced when shorter fragments are rejoined during repair, are not included

in the analysis. To evaluate the contribution of these ‘lost fragments’ we have to

find ways to measure much longer fragments, but also a detailed theoretical

38

modelling of DNA size distributions following high-LET would be helpful

(Sachs et al., 1998).

The tendency toward slower repair as LET values increase, evident in Table 6, is

clearly visualized in Figure 9, where the 80 and 225 keV/µm data are

compared. It can be concluded from the figure that neither the initial number of

fragments, nor their initial size distribution, can explain the obvious slower

repair at higher LET values. The initial level of fragmentation (t=0) below 2

Mbp was the same, or even higher, for the 80 keV/µm ions, compared with the

higher LET. After 4 hours of incubation for DNA repair the situation was the

reverse. Altogether, the results presented here imply that increased DSB

complexity is the determining factor for the reduced repair capacity seen after

exposure to high-LET radiation.

10-8

10-7

100 1000

80 keV/µm, 0h80 keV/µm, 4h225 keV/µm, 0h225 keV/µm, 4h

fract

ion

of D

NA/

δM

(bp-1

)

Fragment size (kbp)

Figure 9. Distribution pattern and rejoining. The fraction of DNA at t=0h and t=4h

was normalized using the width of each size interval (gel segment) and plotted

versus fragment size for 80 and 225 keV/µm nitrogen ions.

(Data from Paper III.)

39

Rejoining and end-points

The prevailing mode for DNA DSB repair in G0/G1 mammalian cells is the

homology-independent end-joining pathway (Wang et al., 1997), a pathway

initiated by the binding of a Ku70/Ku86 protein complex to the DNA ends,

followed by association of the DNA-dependent protein kinase catalytic subunit

(DNA-PKcs). The Ku86 protein is closely related to the fast kinetics (Okayasu

and Iliakis, 1994). However, the biphasic rejoining (see Figure 8) suggests that at

least one additional DNA repair pathway is present, probably representing a

slower process. The present work presents evidence nominating the complexity

of breaks as the determining factor for reduced reparability. The increasing

fraction of DSBs rejoined by the slow phase, together with the elevated level of

remaining DSBs measured after irradiation at high-LET values, could reflect the

failure of the rapid repair processes to rejoin such complex lesions, thereby

increasing the likelihood of misrejoning events. However, recent studies on

misrejoining in a 3.2 Mbp restriction fragment did not show any significant

change with LET (Löbrich et al., 1998). In contrast, mutation analyses have

shown an LET dependency with increased yields for partial or total deletions

ranging from about 5–45 kbp at the HPRT locus for high-LET (Amundson et al.,

1996; Metting et al., 1992). Biophysical modelling also predicts more total

deletions of sizes below 500 kbp (Wu et al., 1998) after high-LET irradiation. It is

conceivable that the fragments produced by correlated breaks within the same

loop domain may be lost in the repair process because they are freely diffusible,

resulting in an increased induction of deletions. Together with data presented

for fragment analysis, this supports the hypothesis that high-LET radiation

damage is related to higher order structures of chromatin. A comparison with

published data on the rejoining of chromosomal fragments as measured by the

premature chromosome condensation technique (PCC) cannot be done as

directly as in the reasoning above. First, the excess of fragments <1 Mbp

observed by fragmentation analysis in the present study cannot be detected by

the PCC technique, and second, a considerable amount of rejoining takes place

during the 20–60 minutes of fusion with the PCC technique. Both these events

lead to an underestimation of the initial number of induced breaks, and will

considerably affect the measured level of unrejoined breaks.

40

Rejoining after exposure to the same number of particles

Complexity of breaks

As regards DSB induction, the rejoining of clustered DSBs after irradiation with

the same number of nitrogen ions traversing the samples was investigated

(Paper IV). The results further supported the complexity of the breaks as the

most important parameter. A slower overall rejoining was found for the higher

LET values tested, with still substantial levels of fragmentation following 1 hour

incubation for repair. For the lowest LET, 80 keV/µm, a major part of the

induced fragments was rejoined already after 30 min (Figure 10).

0

0.2

0.4

0.6

0.8

1

100 1000

0 h0.5 h1 h4 h20 h

80 keV/µµµµm

30 100 1000

175 keV/µµµµm

30

Fragment size (kbp)

Frac

tion

of D

NA/ δδ δδ

M (a

rbrit

ary

units

)

Figure 10. Fragmentation and repair. Fragment size distributions as a function of

mean fragment size after rejoining 0–20h. The cells were irradiated with 5

nitrogen ions per µm2 at 80 and 175 keVµm. Both curves representing the

initial damage (t=0) were normalized by setting the maximum values to unity.

(Data from Paper IV.)

When studying the fragment distributions following different repair times, it

was found that although the smaller fragments were rejoined relatively quickly

even for the 175 keV/µm ions, the number of fragments larger than 2 Mbp did

not change significantly during the first hour of repair at the higher LET. An

alternative explanation for the slower rejoining at higher LET, besides the

increased complexity of the breaks, could be the fact that more breaks are

induced at higher LETs, and that the cellular repair mechanism needs more

time to process all these breaks, due to saturation. However, the results

described above (Paper III) showed that for the same initial number of breaks,

41

and with similar fragment size distributions, those induced by the higher LET

rejoined more slowly. A complementary explanation could be that the slower

rejoining is caused by DSBs induced within compact chromatin structures. Such

breaks might be produced predominantly by direct ionizations as from

high-LET particles, and if there is limited access for repair enzymes/proteins to

such condensed structures, this could result in a reduced capacity of repair

(Ahnström et al., 2000). More experimental work is needed to confirm (or

exclude) this.

Chromatin organization

There is both theoretical and experimental evidence that chromatin

organization on the sub-kilobasepair level, e.g. from nucleosomal winding of

DNA in the 30 nm chromatin fibre, plays a significant role in the induction of

very short DNA fragments (≤2 kbp) — sometimes called “Rydberg fragments”

— for both low and high-LET radiation (Holley and Chatterjee, 1996; Rydberg,

1996). Correlated breaks over much larger distances are also expected due to

the high compaction of the genomic DNA. The packing of DNA is believed to

involve chromatin loops attached to a nuclear matrix (Bodnar, 1988). However,

the possible influence on DNA fragmentation of higher orders of genomic

organization in interphase cells has not been fully elucidated in the literature. It

is possible that such influence could be detected by investigating fragmentation

patterns within a wide molecular weight range, induced by radiation of varying

quality.

When charged particles of the same type (but with differing ionization density)

pass through matter, they show different patterns of energy deposition around

the primary track, due to differences in kinetic energy. The higher velocity of

the ions having lower LET values generates comparatively long-range

secondary electrons, δ-electrons; consequently the energy is distributed more

sparsely throughout the genome. In the present study it is suggested that these

differences in track structure, in combination with target structures, e.g. loop

domains, did influence the yield as well as the size distribution of the induced

damage.

42

Substructures due to chromatin organization?

As illustrated in Figure 11 (left panels), the high-LET ion irradiation tilted the

fragment size distributions in favour of smaller fragments. Furthermore,

substructures were introduced in the distributions, that were not expected from

random breakage (dotted lines). These substructures, or ‘peaks’, were found at

fragment sizes in the range 50–200 kbp and 0.5–1.5 Mbp. As mentioned, the

interphase chromatin is believed to be folded into loops. The exact sizes of these

loops have not yet been accurately characterized, but some evidence of

Mbp-sized loops does exist (Sachs et al., 1995; Yokota et al., 1995). Also smaller

loops, in the range 10–200 kbp, have been suggested in the literature (Bodnar,

1988; Filipski et al., 1990; Jackson et al., 1990; Vogelstein et al., 1980). The peak

structures observed in the present work could be the result of spatially

correlated and clustered breaks within such loops. To isolate the role of

high-order chromatin loops from correlated breaks originating from random-

coiled structures of the genome only, as predicted by some theoretical works

(Hutchinson, 1996; Sachs et al., 1995), detailed analysis of fragmentation

following high-LET irradiation of e.g. naked DNA could be performed, but such

investigations were beyond the scope of this work.

43

0

1 10-7

2 10-7

3 10-7

4 10-7

100 1000

a) 60Co photons

-3 10-7

-2 10-7

-1 10-7

0

1 10-7

2 10-7

100 1000

exp - rand 8 DSB bp-1 Gy-1

exp - rand 6 DSB bp-1 Gy-1

0

1 10-7

2 10-7

3 10-7

100 1000

b) 125 keV/µµµµm

-3 10-7

-2 10-7

-1 10-7

0

1 10-7

2 10-7

100 1000

exp - rand 8 DSB bp-1 Gy-1

exp - rand 6 DSB bp-1 Gy-1

exp - rand 3 DSB bp-1 Gy-1

0

1 10-7

2 10-7

3 10-7

4 10-7

5 10-7

6 10-7

100 1000

c) 225 keV/µµµµm

-3 10-7

-2 10-7

-1 10-7

0

1 10-7

2 10-7

100 1000

exp - rand 8 DSB bp-1 Gy-1

exp - rand 6 DSB bp-1 Gy-1

exp - rand 4 DSB bp-1 Gy-1

Frac

tion

of D

NA/ δδ δδM

(bp-1

)

Non-

rand

om c

ompo

nent

(bp-1

)

Fragment size (kbp)

Figure 11. Fragmentation patterns. The fraction of DNA as a function of fragment

size for (a) 150 Gy 60Co-photons, (b) 100 Gy 125 keV/µm nitrogen ions, and

(c) 200 Gy 225 keV/µm nitrogen ions. Dotted lines represent theoretical

distributions assuming corresponding doses and random breakage, with the

DSB yields 6 and 8 × 10-9 DSB bp-1 Gy-1 (panel a), 3, 6 and 8 × 10-9 DSB bp-1

Gy-1 (panel b) and 4, 6 and 8 × 10-9 DSB bp-1 Gy-1 (panel c). Corresponding

panels (right) illustrate the non-random component of the fragment

distributions (see main text for details). (Data from Paper IV.)

44

Even though not pronounced, a small peak around 100 kbp was seen also for

the photons, indicating that a limited but significant non-random contribution

was present. Such single-hit behaviour, producing smaller DNA fragments

(<1 Mbp), has been reported for low-LET X-ray (Löbrich et al., 1996; Newman et

al., 1997; Rydberg, 1996) and high-energy electrons (Sak et al., 1996).

In Figure 11 (right panels), the non-random contribution to the breakage

patterns is illustrated by subtracting the random curves suggested in the left

panels (from the experimental data). From the appearance of the resulting

fragment size distributions, a close relation to LET is evident regarding the

magnitude of the non-random component, but involvement of higher orders of

DNA folding is also indicated. Due to lower velocity, a particle having a higher

LET value deposits more of its energy by ionization in the direct vicinity of its

path than do particles at a lower LET, which increases the likelihood of

correlated breaks within loop domains, and this may explain the increased non-

random shape of the curves. Thus, the random yield may decrease with

increasing LET, while the number of associated breaks increases. The random

yields chosen in Figure 11 may be too high, as some published data suggest a

formation of more than two additional DSBs for each randomly induced break,

due to clustering of breaks within a few Mbp for high-LET radiation (Sachs et

al., 1998). In fact, the model presented by Sachs et al. 1998 yielded an average

multiplicity of 4 breaks, i.e. 1 random break and 3 non-random breaks, in each

cluster when tested on published data for ions at ~100 keV/µm (Löbrich et al.,

1996; Newman et al., 1997). The same multiplicity, if applied to ion data

presented in Paper II (see Table 4) would give a random yield of around 2–2.5 ×10-9 DSBs bp-1 Gy-1.

This yield is similar to the one used in paper I, where a very simple theoretical

description of this phenomenon was presented. The dose response for the

amount of DNA smaller than 6 Mbp that was released into the electrophoresis

gel was reconstructed, and a close agreement with experimental data was

achieved by applying a relatively low random yield (3 × 10-9 DSBs bp-1 Gy-1) in

combination with additional breaks within 2 Mbp (Paper I). Naturally, in order

to fully isolate the random from the non-random component of the DNA

fragmentation, and to elucidate the relation to chromatin organization, far more

elaborate examinations and detailed theoretical modelling will need to be

performed; this was beyond the scope of the present work.

45

SUMMARY AND CONCLUSIONS

A substancial proportion of non-randomly induced DNA double-strand breaks

was evident when large radiation–induced DNA fragments were analyzed by

pulsed-field gel electrophoresis (PFGE) following exposure to 125 keV/µm

nitrogen ions in the dose-range 0.5–30 Gy. This was in contrast to results for60Co-photons. The non-random contribution to the fraction of DNA released

into the gel may be explained by introducing one or several correlated breaks

within 2 Mbp from a randomly induced break.

The significant non-random contribution to the number of radiation-induced

DNA DSBs for high-LET irradiation, was further confirmed by detailed

fragmentation analysis after exposing normal human fibroblast cells to nitrogen

ions in the LET range 80-225 keV/µm. The radiation quality had a strong

influence on the initial DNA fragment distribution, with an elevated number of

small and medium-sized fragments (5 kbp – 1 Mbp) following high-LET

irradiation. When the fragment size distributions were taken into account, the

ion yields (DSBs bp-1 Gy-1) were some 80–110% higher than those calculated

when disregarding the distributions, i.e. assuming random breakage (FAR

assay). For gamma irradiation the corresponding increase were 13%, and

consequently, the relative yields for DSB induction increased to values

significantly above unity (1.2–1.5) for the high-LET irradiation, however

decreasing for increasing LET values. On the other hand, the relative yields

assessed by conventional assay did not exceed unity for any of the LETs tested.

The DNA fragmentation pattern, i.e. how the DNA DSBs are distributed along

the chromosomes, showed to have an influence on the DSB rejoining in normal

human fibroblasts. DNA fragment analysis detected LET dependent differences

in the amount of residual damage following 20h of incubation for repair, while

no influence of LET on the half-times of the biphasic repair kinetics was found.

In contrast to earlier investigations, the data presented here show a significant

proportion of repair by the rapid phase also for high-LET irradiaion. In fact, the

majority of breaks were restored by the rapid phase for all radiation qualities

tested. However, the fraction of breaks rejoined by the slow phase were closely

related to the radiation quality, with a five-fold increase for the highest LET

values investigated compared to the sparsely ionizing gamma photons. The

46

present work presents evidence nominating the complexity of breaks as one

important determining factor for reduced reparability reported following

high-LET exposure.

The number of breaks induced per particle traversal through a cell nucleus was

calculated for nitrogen ions in the LET range 80–225 keV/µm, boron ions at 40

and 80 keV/µm, and neon ions at 225 and 300 keV/µm. A close relation to LET

was evident, with a continously growing efficiency for fragment induction

when ions pass the cell nuclei at increasing LET values. A nitrogen ion at 225

keV/µm induced more than twice as many DSBs on its passage through the cell

nucleus, compared to an 80 keV/µm ion, and the elevated efficiency for the

higher LET value is probably due to differences in particle track structure. This

was further supported by the result on the heavier neon ions, having similar

LET value (225 keV/µm), which showed a decreased effectiveness for DSB

induction, compared with the nitrogen ions. Furthermore, the shape of the

induced DNA fragment size distributions, following the nitrogen ion exposure,

was highly correlated to the LET, showing sub-structures that may reveal a role

of the higher order of chromatin organisation in the induction and rejoining of

radiation-induced DNA damage.

Data presented in this thesis provide strong evidence for a non-random

induction of DNA DSBs in mammalian cells following high-LET irradiation,

with a probable relation to correlated breaks within the genomic structure.

Futher, the presented work clearly demonstrates the importance of including

detailed information about the fragment distributions in the analyses.

It is believed that data on initial fragmentation and subsequent rejoining, like

the data presented here, may serve as useful input to biophysical modelling

trying to explain the processes causing severe radiation-induced biological

effects. Moreover, such data can increase the general understanding of initial

processes causing radiation-induced DNA damage, and the mechanisms

leading to its restoration.

47

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56

ACKNOWLEDGEMENTS

“Some folk are wise, and some are otherwise.”

(Tobias George Smollet, 1721–1771)

The studies presented in this thesis were carried out at the Division of

Biomedical Radiation Sciences, Uppsala University, with the main financial

support from the Swedish Cancer Society. A large number of people have

contributed in various ways to this work and I want to express my deepest

gratitude to you all.

I am greatly indebted to my supervisor, the “Rising Star” Ph.D. Bo Stenerlöw for

excellent guiding. I am truly impressed by your knowledge and your working

capacity. Further, I want to thank you for being patient during my frequent

periods of indolence…

My co-supervisors:

Professor Jörgen Carlsson, for providing a positive, open and stimulating

atmosphere at BMS. I also thankfully accepted the very few sets given away on

the badminton course. My ego needed that!

MD, Ass. Professor Erik Blomquist, not only for financial support and useful

comments on manuscripts, but also for fruitful “high-LET” meetings and joyous

acclamations.

Mina vänner & kollegor:

Peter Frisk och Anna Sjöström, mina rumskamrater under mina sista dagar på

BMS. – Tack till er båda för att ni inte knorrat då jag spridit ut mig över hela

vårt rum de senaste 6 månaderna…eller var det därför du flyttade över till

onkologkorridoren Anna….?

….och Peter, jag hoppas att du kan uppehålla den gamla goda och hederliga

“little-NASDAQ” känslan som vi grundade! Hav tröst! MultiQ kommer te’baks!

Semi-seniorerna; Lars Gedda and Pär Olsson.

Lars, för din värdefulla hjälp under min korta historia som Mac-användare,

utan din godhet hade mitt förhållande till Mac-världen varit än mer

komplicerat. Pär, jag hoppas vi snart ses över en sip Isle of Arran — Slainte!

57

Nina Tilly, jag har alltid uppskattat glada middagar och fräscha Gingroggar i

goda vänners lag. Dessutom vill jag tacka för din hjälpsamhet då jag ställt

dumma dosimetrifrågor.

Torbjörn Hartman — BMS store pedagog — vill jag tacka för den fina

illustrationen (Figure 2 i avhandlingen).

To all members of INRA — for adopting me into your mysteries…

Mark Lubberink — aldrig omöjlig när det gäller dator och nätverksinsatser.

Alltid “a nice guy” som dessutom befriade mig från min gamla bil. – Tack och

lycka till…!!

Anna and Vladimir for your friendship and hospitality. I thank you both also for

the frequent delivery of Russian Beluga…

Maria Östh-Eklind, tack för att du återställt ordningen, och för att du alltid varit

hjälpsam.

Veronica Asplund Eriksson, Agneta Johansson och Ulla Johansson, för all hjälp med

praktiska detaljer på labbet.

Tack även till alla er andra som jag mött under min tid på BMS — personal,

seniorer, doktorander och alla andra — en fascinerande skara människor.

Alla berörda på The Svedberg Laboratoriet är också värda ett tack, för alla de joner

ni stressat upp åt mig och sedan levererat vid den biologiska bänken.

Jag vill även tacka mina högt värderade vänner från “andra sidan”, det vill

säga från den riktiga världen utanför den ibland inskränkta forskningsdito;

Staffan “Mäklarn” Dufbäck och hans Anna-Karin, jag uppskattar verkligen allt tok

vi hittat på genom åren. Jag är säker på att nya upptåg väntar runt hörnet…

Mikael Ludvigsson, utan din hjälp hade mitt handicap aldrig nosat på

singel…snart ger jag dig en match – slagspel, scratch – “man to man”.

58

Min familj:

Utan ert starka och kärleksfulla stöd hade detta aldrig lyckats.

Mamma och Pappa, min bror Hans och hans fru Katarina och deras härliga

ungar – Ludvig och Elin.

Stort tack till “Kumla—Eriksbergsgänget” för att ni lånat ut er dotter/syster till

mig, och för att ni så gästvänligt inkluderat mig i er familj.

Slutligen – men absolut viktigast – vill jag tacka min alldeles egna Johanna.

Min älskade fru och min allra bästa vän. För ditt oändliga engagemang och din

aldrig sinande positiva livsyn – och för att du aldrig kallar mig “Skrubbilubben”

när nå’n hör….