International Journal of Radiation Biology

ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20

Simultaneous Measurement of Cell Cycle Phase

Position and Ionizing Radiation-induced DNA
Strand Breakage in Single Human Tumour Cells
Using Laser Scanning Confocal Imaging

P.J. Smith & H.R. Sykes

To cite this article: P.J. Smith & H.R. Sykes (1992) Simultaneous Measurement of Cell Cycle
Phase Position and Ionizing Radiation-induced DNA Strand Breakage in Single Human Tumour
Cells Using Laser Scanning Confocal Imaging, International Journal of Radiation Biology, 61:4,
553-560

To link to this article: http://dx.doi.org/10.1080/09553009214551331

Published online: 03 Jul 2009.

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 INT . J. RADIAT . BIOL ., 1992, VOL . 61, NO . 4, 553-560
Technical Note
Simultaneous measurement of cell cycle phase position and
ionizing radiation-induced DNA strand breakage in single human
tumour cells using laser scanning confocal imaging
P. J . SMITH*t and H. R . SYKESt
Downloaded by [University of California, San Diego] at 10:36 03 February 2016
(Received 11 June 1991; final revision received 25 November 1991 ; accepted 27 November 1991)
Abstract. Techniques for the assessment of DNA damage
and repair in individual cells are pertinent to several areas of
research, in particular the study of the heterogeneity of tumour
cell populations in response to anticancer agents . We describe
an adaptation of an in situ alkaline denaturation assay per-
formed on individual nuclei of lysed cells, termed nucleoids,
trapped within an agarose film . A novel aspect of the technique
described in the application of confocal laser scanning fluores-
cence microscopy for the measurement of nucleoid relaxation in
response to DNA damage . The volumes of spherical nucleoids
and their relative DNA contents were determined by ethidium
bromide staining and the analysis of confocal sections through
the equatorial planes of the nucleoids . Mean nucleoid volume
increased as a linear function of X-ray dose (0 . 5-8 Gy) adminis-
tered to intact cells prior to lysis . We provide evidence of
heterogeneity, in asynchronous cultures, in the DNA unfolding/
unwinding characteristics of cells irrespective of cell cycle age .
Bivariate plots of relative DNA content versus nucleoid volume
allowed the direct assessment of cellular repair capacity with
respect to cell cycle position .
1. Introduction
Experimental procedures for the detection of
DNA strand breaks play major roles in several areas
of research whether the element under study is the
damaging characteristics of a given agent or the
responses of a specific biological system to DNA
damage . Strand interruptions and conformational
rearrangements of the higher-order structure of
DNA can arise as consequences of the direct and
indirect effects of chemical and physical agents . Such
changes may also arise as a consequence of DNA
repair (e.g. endonucleolytic incision) . Furthermore,
DNA strand breaks may be generated during analy-
tical procedures (e.g. cleavage at alkali-labile apuri-
nic sites or at drug-stabilized topoisomerase I1-DNA
*Author for correspondence .
tMRC Clinical Oncology and Radiotherapeutics Unit,
MRC Centre, Cambridge CB2 2QH, UK .
0020-7616/92 $3 .00 (0 1992 Taylor & Francis Ltd
complexes), providing a useful monitor for the levels
of the progenitor lesions .
Analysis of the enhancement by strand breakage
of DNA unwinding under alkaline conditions, is the
basis of several assays for DNA damage (review :
Ahnstrom 1988), having the advantages of rapidity,
simplicity and the ease with which such assays can
be adapted for fluorometry rather than primarily
relying upon scintillometry for DNA detection . Nuc-
lear DNA unfolding methods (Cook and Brazell
1975, Ostling and Johanson 1984) have the advan-
tage that several types of DNA lesions may affect
relaxation rates of higher-order structures in depro-
teinized nuclei (Smith et al. 1986a) providing a
means of quantifying the levels of DNA damage
induced by a given agent . Several fluorescence-
based techniques have been established to examine
the unwinding-unfolding of nuclear DNA in indi-
vidual nuclei . Such an approach permits the exam-
ination of population heterogeneity for either the
induction or repair of DNA lesions (Ostling and
Johanson 1984, Roti Rod and Wright 1987, Singh et
al. 1988, Thomas and Thomas 1989, Olive et al.
1990a,b) . We have sought to develop an approach
which would permit correlation of cell cycle phase
position with DNA damage levels in individual cells .
The DNA unfolding method adopted here com-
bines elements of the 'nucleoid halo' method (Rod
Rod and Wright 1987, Thomas and Thomas 1989,
Jackson and Cook 1985) with the conditions devel-
oped for the sensitive detection of DNA damage in
single cells by the micro-gel electrophoresis of nuc-
leoids (or `comet' assay) under alkaline conditions
(Singh et al. 1988) . Nucleoid halos are tangles of
loops of free DNA that emanate from the remnants
of deproteinized nuclei . DNA strand breakage
results in expansion of these halos, providing a
means of quantifying DNA lesion frequency with
detection limits around 5000 breaks per nucleus
 55 4 P. J . Smith and H. R . Sykes
containing 5 x 10 9 bp. The comet assay relies upon
the lysis of individual nuclei embedded within an
agarose matrix and subjecting the residual nuclear
bodies to electrophoresis such that relaxed or broken
DNA is drawn towards the anode and can be
visualized by fluorescence microscopy following
staining of DNA with ethidium bromide . Under
alkaline conditions the comet assay provides a sensi-
tive means of detecting the consequences of DNA
strand break induction in cells exposed to agents
such as X-radiation, hydrogen peroxide or topoiso-
merase II poisons (Singh et al . 1988, Olive et al.
1990b) .
The current approach depends upon four simple
Downloaded by [University of California, San Diego] at 10:36 03 February 2016
propositions : (1) that nucleoids trapped within an
agarose film, in the absence of electrophoresis, would
assume an effectively spherical shape such that nuc-
leoid volume could be determined from diameter
measurements; (2) that the extent of damage-
induced unwinding/relaxation of DNA would be
reflected in the increase in the volume occupied by
DNA released from a nucleoid ; (3) that accurate
fluorometric assessment of total cellular DNA con-
tent of a lysed nucleus would imply cell cycle posi-
tion ; and (4) that the accurate measurement of
fluorescence intensities and nuclear diameters could
be attained using the unique optical sectioning
ability of the laser scanning confocal fluorescence
microscope .
The essential feature of confocal imaging of fluor-
escent specimens is that light from out-of-focus
regions is eliminated from the detection system
(White et al . 1987) . Thus only regions of the speci-
men within a narrow depth of focus are imaged . An
image can be built up, with appropriate filtration for
background noise, of fluorescence from within a
defined volume of the specimen as determined by the
size of the confocal aperture within the optical array .
Electronic processing of the stored image provides an
accurate measure of fluorescence inensity and its
two-dimensional distribution within the optically
sectioned volume . Indeed, by altering the plane of
focus, a series of images can be accumulated and
their projections used to recreate a three-
dimensional image representing the distribution of
fluorescence intensity throughout a specimen .
2. Materials and methods
2 .1 . Cell culture, X-irradiations and flow cytometry
An extablished small cell lung cancer cell line,
NCI-H69/P (designated H69 ; kindly provided by
Dr P . R . Twentyman, MRC Centre, Cambridge)
was grown under standard suspension culture condi-
tions . The drug etoposide (VP16-213 ; Vepesid ;
Bristol Myers Pharmaceuticals, Syracuse, NY) was
provided as 34 mm stock solutions . Irradiations were
performed on intact cells using a 250 kV X-ray
machine (Pantak, Windsor, UK) operating at
15 mA with filtration of 2 . 32 mm of copper half-
value thickness and a dose-rate of 0 .685 Gy/min .
Reference cell cycle distributions were obtained by
flow cytometric analysis using the Cambridge MRC
dual laser flow cytometer incorporating an Innova
70 argon laser (Coherent) tuned to 488 nm . Signals
for forward and 90° light scatter and fluorescence
above 630 nm were collected for 104 cells using the
90° light scatter parameter as the master signal as
described previously (Smith et al . 1985) . Briefly, cells
were permeabilized with Triton X-100, digested
with ribonuclease A (0 .5 mg/ml) and stained with
ethidium bromide (50µg/ml) for 10 min prior to
analysis . DNA fluorescence distributions were ana-
lysed by flow cytometry using a cell cycle phase-
fitting program which assumes normal distributions
for G 1 and G 2/M phase populations . A probability
function is calculated for the S phase distribution
based upon the means and standard deviations of
the G 1 and G2/M phases (Watson et al. 1987) .
2.2 . In situ lysis and alkaline denaturation of nuclei in
agarose films
The original microgel electrophoresis method
used a neutral lysis solution containing detergent
(Ostling and Johanson 1984) while more recent
adaptations (Singh et al . 1988, Olive et al . 1990a)
have employed alkaline conditions . The basic tech-
nique adopted was that described by Singh et al .
(1988) . Briefly, single sample preparation was as
follows . A thin layer of agarose was prepared on a
microscope slide (Dakin fully frosted, Labcraft
Brand ; Curtin Matheson Scientific, Inc ., Houston,
TX) by placing a large glass coverslip over a 85µl
volume of 0. 5% normal melting point agarose (pre-
pared in filtered phosphate buffered saline, PBS, and
held at 45°C) and placing the slide on a metal plate
cooled to ice temperature . Cells were resuspended in
85 µl of PBS in pre-warmed vials and mixed with
85µl of 1 % low melting point (LMP) agarose and
cells dispersed by vortexing . The coverslip was
removed from the first layer of agarose and 75 µl of
LMP agarose containing cells (5 x 10 4 cells/slide)
dispensed on top of the first layer and again spread
by applying a coverslip and allowing to set for 5 min .
A final layer of 75µl 0 .5% LMP agarose was used to
cover the layer containing cells by repeating the
 DNA damage and cell cycle
above procedure . At this point cells were X-
irradiated at ice temperature, coverslips removed
and cells lysed by immersing slides for 1 h in a lysing
solution (2 .5 M NaCl, 100 mm EDTA, 10 mm Tris, pH
10.0, 1 % sodium sarcosinate supplemented with 1
Triton X-100 and 10% DMSO prior to use) . At this
stage, slides would normally be transferred to an
electrophoresis tank containing an alkaline buffer
(0.3 M NaOH, 1 mm EDTA) and the gels allowed to
equilibrate for 40 min at 15 °C prior to electrophore-
sis for 24 min at 20 V and 300 mA . In the current
study duplicate slides were produced permitting the
comparison of electrophoresed and sham-
electrophoresed samples . Neutralizing buffer (0-4m
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Tris, pH 7 .5) was then gently pipetted onto each
slide (electrophoresed or non-electrophoresed sam-
ples) for 5 min, then the procedure was repeated
before draining and adding 7511 of ethidium bro-
mide solution (2 pg/ml) to the centre of the slide and
replacing the coverslip to spread stain over whole
slide . Slides were stored at 4°C in the dark and in a
humidified atmosphere prior to analysis . Samples
were examined within 24 h to minimize the effects of
specimen compression due to contraction of the
agarose layers upon drying .
2.3 . Confocal laser scanning microscopy
The confocal imaging system used was a BioRad
MRC-600 (BioRad Microscience Ltd, Hemel
Hempstead) scanning assembly incorporating a
25 mW argon ion laser and coupled to a Nikon
Optiphot II fluorescence microscope . Excitation was
at 514 nm wavelength and the scanning beam inten-
sity was reduced to 3% of maximum by neutral
density filtration . The objective lens gave a 20x
magnification (NA=0 . 5) and the digitized image
was accumulated under Kalman filtration to reduce
the signal-to-noise ratio . The system was operated at
its minimum confocal aperture, collecting fluores-
cence from 0 .6µm deep disc-shaped sections of each
nucleoid . Using digitized images the volume of a
spherical lysed nucleus could be determined from the
mean of two diameter measurements for the equa-
torial disc . The DNA content was estimated by
correcting the integrated fluorescence intensity of
the equatorial disc by the factor 2 . 22 x radius (i .e.
the ratio of the nuclear volume to the approximated
disc volume, 1 .331rr 3/0-67rr 2 ) . Routinely, successive
disc images from 40 to 180 randomly selected nu-
cleoids, for each treatment condition, were stored on
optical cartridge for subsequent analysis . Pixel inten-
sity bands were colour-coded to aid both in the
selection of appropriate photomultiplier gain and
555
black level settings and in the analysis of the dis-
played images . The edge of the nucleoid was defined
by the contour at 1 % of the maximum pixel inten-
sity obtained for control nucleoids .
3. Results
3.1 . DNA image cytometry
Optical sectioning (Townsend et al. 1990) of
freshly prepared samples demonstrated that nu-
cleoids trapped in agarose were essentially spherical
(data not shown), permitting the identification of
central sections showing the maximum nuclear dia-
meters and the determination of DNA content as
described above. Initially we sought to determine
whether confocal imaging could reflect the cell cycle
distribution patterns of human tumour cell popula-
tions independently characterized by flow cyto-
metry . An example of such a comparison is shown in
Figures 1 a and 1 b . Asynchronous H69 cultures were
compared with a perturbed population of cells
exposed to the anticancer DNA topoisomerase II
poison VP 16-213 (2µM for 24h) . Conventional flow
cytometry, analysing up to 1 x 10 4 drug-treated
cells, clearly shows the expected drug-induced
reduction in the G 1 fraction and accumulation in
late S and G2 phases (Smith et al. 1986) . Figures Ia
and 1 b also show the distribution of DNA contents in
parallel samples examined by confocal microscopy .
Despite only 100 cells being analysed for each sam-
ple, the DNA distributions were similar to those
obtained by flow cytometry .
3.2. Analysis of nucleoid unwinding/unfolding by confocal
imaging
Figure 2 shows examples of images of sectioned
nucleoids derived from control and irradiated cell
populations . Irradiation causes an increase in the
diameter of the nucleoid body, whereas a I h postir-
radiation recovery period results in a return to an
essentially normal configuration . Figure 3 shows
scans of pixel intensities across representative nu-
cleoid bodies . A lower threshold of 1 % maximum
pixel intensity was used to determine the disc dia-
meter and thereby calculate the nucleoid volume .
Figure 4 indicates that the mean values for nucleoid
volume increase as a linear function of X-ray dose
for the range of 0 . 5-8 Gy. Calculating a Student's
t-value for the correlation coefficient yields ap value
of <0-01 . The large standard deviations shown in
Figure 4 indicate the spread of nucleoid volume
 556 P. J. Smith and H. R . Sykes
values for an asynchronous, total cell population .
This spread is in part due to the increase in absolute
nucleoid volume as a function of increasing cell cycle
age, for both control and irradiated populations (see
Figures 6a and 6b, below) . The differences between
the mean values for irradiated and unirradiated
populations were significantly different when the
results of independent determinations were com-
pared . This was analysed by assuming a null
hypothesis and assigning a value of unity to the
mean nucleoid volume of the control population .
The mean value of [irradiated nucleoid volume/
mean control nucleoid volume] was calculated for 50
Downloaded by [University of California, San Diego] at 10:36 03 February 2016
individual, irradiated (4 Gy) nucleoids and the data
compared for eight separate experiments (mean
ratio 2-16±0-15 SE) each incorporating its own
control sample . Using a one-group (two-tail) t-test
the mean ratio values were found to be significantly
different from controls (p<_ 0-000 1) .
120
a
100
80
c
m
. 60
m The bivariate displays in Figures 6a-c indicate
u- 40
20
0 diation incubation period . This finding is consistent
0 200 400 600 800
Relative DNA content
120
100
80
1 60
40
20
0
200 400 600 800
Relative DNA content
Figure 1 . Frequency distributions of nuclear DNA contents
for control (a) and growth-perturbed (b) H69 cell
populations . Perturbed populations were generated by
exposing cells to 2 µpa VP16-213 for 24h . Distributions
were determined by confocal microscopy (squares ; 100
cells analysed) or by flow cytometry (bold line ; 1 x 10°
cells analysed ; G 1 phase=42 .0% and 2 . 9%, S phase
=36 .0% and 63 . 3%, G 2 /M phase=22 .0% and 33 . 8%
for control and perturbed populations respectively) . Left
and right arrows indicate the positions of G 1 and G 2 /M
phases respectively . Relative DNA content is given in
arbitrary units .
3 .3 . Nucleoid unwinding/unfolding as a junction of cell
cycle position
The calculated DNA contents of sectioned nu-
cleoids of asynchronous H69 cells were ranked and
nucleoids allocated to G 1 , S or G2 /M phases accord-
ing to the expected proportions indicated by parallel
flow cytometric analysis . Figures 5a-f show the
effects of acute X-irradiation (4 Gy) on the nuclear
volumes as a function of cell cycle age . There is
heterogeneity in the radiation-promoted relaxation
responses of nucleoids in all cell cycle phases, par-
ticularly G2/M phase cells . Irradiation resulted in
increases in the mean volumes by factors of 1 .64,
2 . 02 and 1 .93 for G 1i S and G 2/M phase nucleoids
respectively . The results suggest that the DNA
unfolding/unwinding characteristics of S and G 2
phase nucleoids (M phase being less than 1 .5% of
the total population) are similar while nucleoids in
G 1 phase appear to show reduced relaxation
capacity .
3 .4 . Repair of X-ray-induced DNA damage as a junction
of cell cycle position
that irradiated H69 cells are capable of recovering
essentially normal nucleoid volume profiles, irres-
pective of cell cycle position, following a 1 h postirra-
with the previously reported rapid repair kinetics for
radiation-induced DNA damage detected by the
comet assay (Singh et al . 1988, Olive et al . 1990a) . In
the present study there was no evidence of non-
repairing subpopulations of H69 cells .
4. Discussion
The present study has indicated the feasibility of
using confocal imaging in the analysis of the
unwinding/unfolding of DNA from nucleoid-like
bodies embedded within agarose, as a means of
indirectly quantifying the level of DNA damage
within single cells . The non-electrophoretic
approach described here provides an alternative
method for the analysis of in situ relaxation/
unwinding, and allows heterogeneity to be corre-
lated with the important feature of cell cycle age .
Such a methodology would be relevant to studies on :
lesions associated with cell cycle arrest, sensitivity of
specific subpopulations (e.g. aneuploid components
of biopsy specimens) to DNA damage, and the
evaluation of drug-induced damage in relation to
 DNA damage and cell cycle 557
Downloaded by [University of California, San Diego] at 10:36 03 February 2016

Figure 2 . Confocal images of optically sectioned nucleoids showing the effects of the induction of DNA damage and its subsequent
repair . Nine randomly selected images are shown for control (a) or irradiated (4 Gy) cells analysed immediately (b) or after 1 h
recovery at 37 ° C (c) . The central image in each set has been enlarged and reproduced below each set (d, e and f) . Scans of
pixel intensities along the x -y axes are shown in Figure 3 .

cell-cycle regulated cofactors or targets such as DNA

topoisomerase II (Heck et al . 1988, Markovits et al .
1987) .
Several factors have provided an impetus for the
15
development described here . The comet assay, as
previously described (Singh et al. 1988, Olive et al . 10
1990a), presents a problem in the quantification of
the degree of relaxation/unwinding and the relative
DNA contents of the `tail' and the `head' of an
electrophoresed nucleus (Olive et al . 1990a) . Simi- 0
0 2 4 6 8
X-ray dose (Gy)

Figure 4. Increase in mean nucleoid volumes as a function of

X-radiation dose . Data represent mean values for 50
nucleoids (± SD) with a simple linear regression fit
(y=1 . 5x+3 . 87 ; r 2 =0 . 993 ;p<0 . 01) .

larly, nucleoid halo expansion methods (Roti Rod

20 40 60 80
Nucleoid diameter (gm)
Figure 3 . Scans of pixel intensities along the x -y axes shown in
Figure 2 d, e and f. Data from control (feint line) or
irradiated (4 Gy) cells analysed immediately (bold line)
or after I h recovery at 37°C (broken line) .
and Wright 1987, Thomas and Thomas 1989)
employ conditions under which accurate determina-
tion of total DNA content per nucleoid body is
difficult .
Our approach essentially adopts the simple
assumptions made for the halo method while
employing conditions which enhance DNA unwind-
ing and maintain the nucleoid body as a regular-
shaped structure . Coupled with this approach is the
 558 P. J. Smith and H. R . Sykes

use of confocal imaging as a method for the accurate
determination of fluorescence parameters with the
advantage of reduced signal-to-noise levels . A rela-
tively simple approach was adopted for determining
the volume-corrected fluorescence intensities of nu-
cleoids . An alternative approach could involve Z-
axis sectioning of nucleoids and the summing of the
integrated intensities of the sections . Likewise
volumes could be determined using the sum of disc
areas over the confocal planes passing through the
nucleoid times the space between sections (Rigaut
et al. 1991) . Data derived from Z-axis sections would
take account of the three-dimensional shape of the
nucleoid and any non-homogeneous distribution of
DNA within that volume . Furthermore, Z-axis sec-
tioning of nucleoid preparations could be used to

20 14
a
Downloaded by [University of California, San Diego] at 10:36 03 February 2016

,8
12
d
16
14 10
12
1= 8
10 0
v 8 0 6
6 4
4
2
2
0 . I 1I fl ° -II
0 2 4 6 8 10 12 14 16 18 20 0 2 4 8 10 12 14 16 18 20

22 .5 7
20 b 6
e
5
15
E 4
0 0
10 0 3

2
5
1
° r 1 rl Il"
0 2 4 6 8 10 12 14 16 18 20 °0 2 4 6 10 12 14 16 18 20

9 5
8 .c f
7 4

6
~3
1= 5
0 0
U4 U 2
3
2 1
1
0 r 0 .
0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
Nudeoid volume x 10 -4 (µm 3) Nudeoid volume x 10 -4 (µm 3)

Figure 5 . Distribution of nucleoid volume values for G 1 (a, d), S (b, e) and G 2 /M (c, f) phase nucleoids . Control (a-c) or
X-irradiated (4 Gy ; d-f) samples. Data derived from an exponentially growing cell population (47% G 1 ; 34% S; 19% G2 /M ;
determined by flow cytometry) ; 134 control and 179 irradiated nucleoids analysed .
 DNA damage and cell cycle
Downloaded by [University of California, San Diego] at 10:36 03 February 2016
a 20
b current technique has been used successfully to
0 0
0 monitor drug-induced DNA damage in small cell
00
0 lung cancer cells, SV40 transformed fibroblasts, hu-
0
0 0 0 8o
0 cg
0 lines (data not shown) . The agents studied to date
00
8nk 0 camptothecin and mitoxantrone .
0
0 10 20 30
DNA content
Figure 6 . Induction and repair of DNA damage as a function
of total cellular DNA content . (a) Control cells, line
fitted by simple linear regression (y=0 . 12x+ 1 .08 ;
r2 = 0 . 815 ; p < 0 . 0001) and reproduced in b and c . (b
and c) X-irradiated (4 Gy) cells analysed immediately
or after 1 h recovery at 37 ° C, respectively.
automatically analyse large numbers of nucleoids
despite the presence of overlapping nucleoids in the
Z-axis direction .
Heterogeneity was observed in the expansion of
nucleoid bodies under alkaline conditions . Such
heterogeneity has been noted previously for `comet'
formation in irradiated nucleoid populations . A pre-
vious report suggested that heterogeneity is not
merely a reflection of cell cycle age as indicated by
experiments using G 1 or G 2 blocked cultures of
559
Chinese hamster cells (Olive et al . 1990a) . The
present study, using asynchronous human tumour
cells, supports that view. Heterogeneity could be due
to different factors according to the cell cycle phase
position. For example, within G 1 phase some H69
cells show low RNA :DNA ratios characteristic of
either Go or G IA phase (Smith et al. 1990), with the
possibility of associated changes in the packing or
unfolding characteristics of chromatin (Smith et al.
1985) .
It is possible that the conditions of the assay could
be optimized further with respect to potentially
important variables such as : pH, lysis time, ethidium
bromide or agarose concentrations . However, the
man adenocarcinoma cell lines and mouse L cell
include the topoisomerase inhibitors VP16-213,
It is common practice to use ionizing radiation as
40
a dosimetric standard for calculating the frequencies
of lesions induced by cytotoxic drugs (Kohn et al.
1980, Smith 1987) . Within the present context such
comparisons would depend upon the assumption
that DNA from irradiated and drug-treated nuclei is
released in a similar form and therefore migrates in a
similar fashion through the agarose matrix . Using
the fast photon counting facility of the MRC-600
system we have examined the substructure of the
weakly fluorescent DNA migrating into the agarose
gel during lysis, or drawn from the nucleus by
microelectrophoresis . We have observed a complex
structure of irradiated DNA within the agarose
matrix, consisting of fluorescent spherical aggrega-
tions each approximately 1µm in diameter, inter-
spersed with groups of aggregations often involving
several units. There was no evidence that the electro-
phoretic mobility or centre-to-periphery distribution
for the single and the multiple units were different .
Similar structures were observed in lysed nucleoids
from VP16-213-treated cells, suggesting that the
unit structure reflects packaging of nuclear DNA
(e.g. chromatin loop domains) rather than being
specifically dependent upon the nature of the strand
break-inducing agent .
We conclude that the current study provides a
basis for an alternative approach to measuring DNA
damage in defined subsets of cells . The increasing
availability and sophistication of confocal micro-
scopes and their associated image analysis routines
should permit the automated analysis of the poten-
tial responsiveness of human tumour cells to anti-
cancer agents .
 560 DNA damage and cell cycle
Acknowledgements
We thank Professor N . M . Bleehen for encourage-
ment and support, Dr J . V. Watson for provision of
the flow cytometry facility and Dr M . Green and
Mrs J . Lowe (MRC Cell Mutation Unit, Sussex) for
advice and demonstration of the single cell micro-
electrophoresis technique .
References
AHNSTROM, G ., 1988, Techniques to measure DNA strand
Downloaded by [University of California, San Diego] at 10:36 03 February 2016
breaks in cells : A review . International Journal of Radiation
Biology, 54, 695-707 .
COOK, P. R . and BRAZELL, I . A ., 1975, Supercoils in human
DNA . Journal of Cell Science, 19, 261-279 .
HECK, M . M ., HITTELMAN, W . N, and EARNSHAW, W . C .,
1988, Differential expression of DNA topoisomerases I
and II during the eukaryotic cell cycle . Proceedings of the
National Academy of Science, USA, 85, 1086-1090 .
JACKSON, D. A . and COOK, P. R ., 1985, A general method of
preparing chromatin containing intact DNA. EMBO
Journal, 4, 913-918 .
KOHN, K . W ., EWIG, R . A . G ., ERICKSON, L . C . and ZWELLING,
L . A., 1980, Measurement of strand breaks and cross
links by alkaline elution . DNA Repair, edited by E . C .
Friedberg and P. C . Hanawalt (Marcel Dekker Inc .,
New York), Vol . 1B, pp . 379-401 .
MARKOVITS, J ., POMMIER, Y., KERRIGAN, D., COVEY, J . M .,
TILCHEN, E . J . and KOHN, K . W ., 1987, Topoisomerase
II-mediated DNA breaks and cytotoxicity in relation to
cell proliferation and the cell cycle in NIH 3T3 fibro-
blasts and L1210 leukaemia cells. Cancer Research, 47,
2050-2055 .
OLIVE, P . L ., BANATH, J . P . and DURAND, R . E ., 1990a,
Heterogeneity in radiation-induced DNA damage and
repair in tumor and normal cells measured using the
`comet' assay . Radiation Research, 122, 69-72 .
OLIVE, P . L ., BANATH, J . P . and DURAND, R. E ., 1990b,
Detection of etoposide resistance by measuring DNA
damage in individual cells . Journal of the National Cancer
Institute, 82, 779-783 .
OSTLING, 0 . and JOHANSON, K . J ., 1984, Microelectrophoretic
study of radiation-induced DNA damages in individual
mammalian cells . Biochemical Biophysical Research Commu-
nication, 123, 291-298 .
RIGAUT, J . P ., VAssY, J ., HERLIN, P., DUIGOU, F., MASSON, E .,
BRIANE, D ., FOUCRIER, J ., CARVAJAL-GONZALES, S .,
DOWNS, A . M ., and MANDARD, A .-M ., 1991, Three
dimensional DNA image cytometry by confocal scan-
ning laser microscopy in thick tissue blocks . Cytometry,
12, 511-524.
ROT! ROTI, J . L . and WRIGHT, W . D., 1987, Visualization of
DNA loops in nucleoids from HeLa cells : assays for
DNA damage and repair . Cytometry, 8, 461-467 .
SINGH, N . P ., McCoy, M . T ., TICE, R . R . and SCHNEIDER,
E . L ., 1988, A simple technique for quantitation of low
levels of DNA damage in individual cells, Experimental
Cell Research, 175, 184-191 .
SMITH, P. J ., 1987, Ferrous iron-mediated enhancement of DNA
damage and recovery potential in bleomycin-treated
human cells . Biochemical Pharmacology, 36, 475-480 .
SMITH, P. J ., ANDERSON, C . 0 . and WATSON, J . V ., 1985, Effects
of X-irradiation and sodium butyrate on cell-cycle tra-
verse of normal and radiosensitive lymphoblastoid cells .
Experimental Cell Research, 160, 331-342 .
SMITH, P . J ., ANDERSON, C . 0 . and WATSON, J . V ., 1986a, A
predominant role for DNA damage in etoposide (VP-
16-213) induced cytotoxicity and cell cycle perturbation
in human SV40 transformed fibroblasts . Cancer Research,
46, 5641-5645 .
SMITH, P . J ., MIRCHEVA, J . and BLEEHEN, N . M ., 1986b,
Interaction of bleomycin, hyperthermia and a calmodu-
lin inhibitor (trifluoperazine) in mouse tumour cells : II .
DNA damage, repair and chromatin changes . British
Journal of Cancer, 53, 105-114.
SMITH, P . J ., MORGAN, S . A ., Fox, M . E . and WATSON, J . V .,
1990, Mitoxantrone-DNA binding and the induction of
topoisomerase II associated DNA damage in multidrug-
resistant small cell lung cancer cells . Biochemical Pharma-
cology, 40, 2069-2078 .
THOMAS, E . A . and THOMAS, C . A ., 1989, Nucleoid halo expan-
sion indirectly measures DNA damage in single cells .
Experimental Cell Research, 183, 149-158 .
TOWNSEND, K . M . S ., STRETCH, A ., STEVENS, D . L . and GOOD-
HEAD, D . T., 1990, Thickness measurements on V79-4
cells : a comparison between laser scanning confocal
microscopy and electron microscopy . International Journal
of Radiation Biology, 58, 499-508 .
WATSON, J . W ., CHAMBERS, S . H . and SMITH, P. J ., 1987, A
pragmatic approach to analysis of DNA histograms with
a definable G1 peak . Cytometry, 8, 1-8 .
WHITE, J . G ., AMos, W . B . and FORDHAM, M ., 1987, Evaluation
of confocal versus conventional imaging of biological
structures by fluorescence light microscopy . Journal of
Cell Biology, 105, 41-48 .