(CANCER RESEARCH 49, 5054-5057, September 15, 1989]
Association among DNA/Chromosome Break Rejoining Rates, Chromatin Structure
Alterations, and Radiation Sensitivity in Human Tumor Cell Lines1
Jeffrey L. Schwartz2 and Andrew T. M. Vaughan3
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois 60637 [J. L. S.], and Division of Biological and Medical Research, Argonne
National Laboratory, Argonne, Illinois 60439-4833, and Department of Immunology, Birmingham University, Birmingham, BI5 277, United Kingdom [A. T. M. V.)
ABSTRACT
The basis for radioresistance and radiosensitivity in human tumor cell
lines is unknown. In a previous study, radiosensitivity in human tumor
cell lines was found to be a function of the rate of DNA double-strand
break rejoining. Radioresistant cell lines rejoined DNA double-strand
breaks at a faster rate than more sensitive cell lines. In this study, we
have expanded on that work and analyzed the rate of chromosome break
rejoining, as well as the type and frequency of chromosome aberrations
induced in three relatively radioresistant (/)„
> 2.0 Gy) human squamous
cell carcinoma cell lines and three relatively radiosensitive (/>„
< 1.5 Gy)
squamous cell carcinoma cell lines. Radioresistant cells were found to
rejoin chromosome breaks faster than more sensitive cells. The faster
rate of rejoining was associated with a reduced frequency of misrepair
events (chromosome exchange-type aberrations) and greater survival.
There were qualitative differences between these two groups of cell lines
in their ability to bind ethidium bromide as nucleoids, suggesting that the
basis for altered break rejoining rates might be related to chromatin
structure.
INTRODUCTION
Ionizing radiation produces a variety of DNA lesions includ
ing single- and double-strand DNA breaks and base alterations
(1). The mechanisms by which cells respond to these lesions
are complex, involving many different enzymatically driven
reactions. Most studies on the molecular mechanisms underly
ing radiation responses in mammalian cells have focused on
the induction and repair of DNA double-strand breaks, because
DNA double-strand breaks are believed to be the primary
radiation-induced lethal lesion (2). Usually, mutant rodent cell
lines which are sensitive in their response to radiation damage,
as compared to the corresponding wild type cell lines, are used.
In these rodent cell lines, radiosensitivity is often a function of
either the initial radiation-induced DNA double-strand break
frequency or the cell's capacity to rejoin DNA double-strand
breaks (3-7). Many of the radiation-sensitive mutant cells fail
to rejoin a significant portion of the radiation-induced DNA
double-strand breaks (5-7).
There is little information concerning the basis for radiosen
sitivity and radioresistance in human cells. In part this is due
to the fact that there are few good human cell models for
radiation sensitivity and resistance. Ataxia telangiectasia is a
radiosensitive genetic syndrome that has been under study for
some time (8). Conclusions concerning the underlying cause of
sensitivity in ataxia telangiectasia cells, however, are not firm.
There are reports of alterations in chromosome break-rejoining
ability, but these findings appear to be dependent on the specific
cell line examined (9, 10).
Received 1/17/89; revised 4/17/89, 5/26/89; accepted 6/15/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the National Cancer Institute (CA
42596 and CA 37435), the Department of Energy (W-31-109-ENG-38), and the
Cancer Research Foundation.
2 Recipient of a Junior Faculty Research Award from the American Cancer
Society. To whom requests for reprints should be addressed, at Department of
Radiation and Cellular Oncology, University of Chicago, 5841 S. Maryland Ave.,
Box 442, Chicago, IL 60637.
3On sabbatical from the University of Birmingham during the course of these
studies.
As a model system for variations in human cellular radiosen
sitivity, human tumor cell lines provide a unique resource.
Human tumor cell lines have been reported to range in sensi
tivity (D0) from as low as 0.7 Gy to more than 3.0 Gy (11-14).
[Normal human fibroblasts range in sensitivity from about 1.2
to 1.8 Gy (15, 16).] Analyzing the induction and repair of
radiation-induced DNA damage in such tumor cells could pro
vide important insights into the mechanisms that underlie not
only radiation sensitivity but also radiation resistance in human
cells. Furthermore, because of the suggestion that the presence
of inherently radioresistant cells in a tumor might underlie
radiotherapy failure (11-13), study of the basis of human tumor
cell radioresistance might suggest predictive assays of tumor
response or alternative therapeutic strategies.
In an earlier study, the induction and rejoining of DNA
double-strand breaks was studied in a group of human tumor
cell lines that ranged in sensitivity from about 1.1 to 2.8 Gy
(17). It was found that, in human tumor cell lines, radiation
sensitivity was associated with a slower rate of DNA doublestrand break rejoining.
These earlier studies involved DNA neutral elution analysis
(18) after exposure to 100-Gy 60Co 7-rays. In the present study,
we have extended these observations and examined chromo
some break rejoining after 3-Gy exposures to X-rays. Numerous
studies have suggested that chromosome aberrations lead to
cell death and that DNA double-strand breaks underlie both
phenomena (19, 20). Chromosome analysis allows for the meas
urement of both unrejoined chromosome breaks (terminal dele
tions) and misrepair events (exchange-type aberrations; dicentrics, rings, and interstitial deletions). In addition, by split-dose
experiments, the time that chromosome breaks remain open
(restitution time) can be estimated (21). As the time interval
between fractions increases, the frequency of exchange-type
aberrations decreases as breaks induced by the first dose of
radiation are rejoined and are no longer available for interaction
with breaks induced by the second dose of radiation.
We also investigated possible factors that might affect breakrejoining rates. One candidate was chromatin structure. Alter
ations in chromatin structure could affect the access of DNA
repair enzymes to DNA strand breaks or the time that DNA
breaks remain in the appropriate configuration for accurate
rejoining of DNA double-strand breaks. To examine chromatin
structure differences, we investigated the ability of ethidium
bromide to intercalate into the DNA of salt-extracted nuclei
(nucleoids) (22, 23). Nucleoids contain the entire nuclear DNA
attached to a protein matrix and they retain the supercoiled
organization of the intact cell.
MATERIALS AND METHODS
Cell Lines and Culture Conditions. Six human squamous cell carci
nomas were studied. These cell lines were established from tumor
biopsies as described previously (11-14, 17). Cells were maintained
under exponential growth in complete medium (72.5% Dulbecco's
modification of Eagle's medium, 22.5% Ham's Nutrient Mixture F-12,
5% fetal calf serum, 0.4 fig/ml hydrocortisone, 100 units/ml penicillin,
and 100 ¿jg/mlstreptomycin).
5054
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RADIOSENSmVITY,
REPAIR, AND CHROMATIN
DNA Double-Strand Break Analysis. The frequency of DNA doublestrand breaks was estimated using the neutral elution assay (17, 18).
Exponentially growing cells were labeled with 0.02 fiCi/ml [14C]thy-
Table 1 Characteristics of human tumor cell lines
The radiation survival parameters /),, and n were determined by least squares
regression analysis (11-14). The chromosome number ±SE was determined from
SO metaphase cells. The DNA index is the G, DNA content normalized to a
diploid human lymphoblastoid cell line. The ACT (average generation time, in
h) was determined from growth curves.
midine for 48 h. Cells were then washed and cultured in nonradioactive
medium for 4 h before being irradiated in ice-cold saline with 100-Gy
""Co 7-rays from a Gamma Cell 220 irradiator (Atomic Energy of
Canada) at a dose rate of 5.25 Gy/s. After irradiation, cells were
incubated for various times in complete medium at 20°Cbefore being
lineSCC-12B.2JSQ-3SQ-20BSQ-9GSQ-38SCC-25Do2.662.632.381.461.451.42nl.l.7.4.4.8.5Chrom
Cell
no.72.4
1.481.4
±
±2.557.6
1.056.7
±
0.581.9
±
±2.069.4
±1.1DNAindex2.022.571.841.812.502.51ACT(h)27.2
scraped off the plates with rubber policemen; elutions were run at pH
9.6 as described previously ( 17, 18). To compare elution profiles, strand
scission factors were calculated as described by Murray et al. (24). This
value corresponds to the relative frequency of DNA strand breaks. It is
defined as log [(.£)//,)],where f, is the fraction of DNA retained on the
filter, after 12-h elution, in the irradiated sample, and /„is the filter,
after 12-h elution, in the non irradiami control. All results are the mean
of at least three separate experiments. The standard errors were always
less than 10% and usually less than 5%. Results are presented as
percentage of DNA damage rejoined as a function of time after irradia
tion.
Chromosome Aberration Analysis. Cells were irradiated in d with
3-Gy X-rays (250 kVp, 20 mA) at a dose rate of 0.8 Gy/min and were
incubated at 37°Cbefore harvest 18-30 h later. Two h before harvest,
2 x IO"7 M colcemid was added to the cultures. Cells were removed by
trypsin treatment and were exposed to 0.075 M KC1for 15 min, followed
by two washes in methanol:acetic acid (3:1). Slides were prepared and
stained with a 5% Giemsa in Gurr buffer solution. Then, 100-200 cells
were counted for each treatment and cell line. Four types of chromo
some aberrations were distinguished: terminal deletions, interstitial
deletions, dicentrics, and rings (and accompanying acentric fragments).
Harvest time was dependent on the average generation time determined
as described below. Autoradiographic analysis of [3H]thymidine-labeled
parallel cultures ensured that all the cells analyzed were in Gt at the
time of irradiation. For the split-dose experiments, cells were irradiated
with two 1.5-Gy (or two 3.0-Gy) X-ray doses that were separated by 0,
1, 2, 3, or 4 h at 37°C.Chromosome content (average chromosome
number/cell) was determined in nonirradiated cells from 50 metaphase
samples.
Cell Cycle Analysis and Determination of DNA Index. For cell cycle
analysis, exponentially growing cells were fixed in 70% ethanol and
stained in 2 ¿ig/ml4,6-diamidino-2-phenylindole (Sigma) and the flu
orescence histogram was accumulated on a Partee PAS II cytometer.
A DNA index was calculated as the ( i, DNA content in the tumor cells,
normalized to a diploid human lymphoblastoid cell line. To determine
the growth kinetics of the cell lines studied, IO5cells in 75-cm2 flasks
that contained 15 ml of complete medium were initiated and cell
number/flask was determined daily over 1-week period. Average gen
eration times were estimated from the exponential portion of the growth
curve.
Nucleoid Analysis. Nucleoids were prepared by suspending 5 x 10s
cells in 0.5 ml of lysis solution that contained 2 M NaCl, 10 HIMTris
buffer, pH 8.0, 10 mM EDTA, and 0.5% Triton X-100 (22). Nucleoids
were stained with ethidium bromide to a final concentration of 20 Mg/
ml and were analyzed after 90 s on an Ortho cytofluorograph II that
was modified to operate with a Becton Dickinson FACS 440 jet-in-air
flow cell system. Data from 10,000 nucleoids were accumulated for
each sample. For each cell line, a cumulative frequency histogram was
constructed of the <.'•,
fluorescence peak. The data are expressed as a
percentage of the total histogram counts, summed at intervals of 10
channels.
RESULTS
The characteristics of the six human squamous cell carcinoma
cell lines studied are shown in Table 1. Radiation sensitivity,
measured in exponentially growing cultures of each cell line,
has been previously reported (11-14). Most of the published
work has been on early passages of these cell lines. Therefore,
radiation sensitivity was reexamined in the much later passage
cell lines used in this study. Only small differences (<10%)
were noted between the earlier studies and those performed
STRUCTURE
30
45
60
75
TIME AFTER IRRADIATION (rr
90
lutes)
105
120
Fig. 1. Percentage of DNA double-strand break damage remaining as a func
tion of time after a 100-Gy exposure to *°Co-y-rays in radioresistant cell lines
JSQ-3 P), SCC-12 B.2 (A), and SQ-20B (O) and in radiosensitive cell lines SCC25 (A), SQ-38 (•),and SQ-9G (•).Curves, mean values (
, radioresistant cell
lines;- •¿
•¿
-, radiosensitivity cell lines).
here. The radiation survival curve parameter Da (radiosensitiv
ity) ranged from 1.42 Gy (SCC-25) to 2.66 Gy (SCC-12 B.2).
The extrapolation numbers (n) were all similar, ranging from
1.4 to 2.1. On the basis of results published for normal human
fibroblasts (15, 16), three cell lines were judged to be relatively
radioresistant (JSQ-3, SCC-12 B.2, and SQ-20B). All three had
Do values greater than 2.0 Gy. The other three were considered
to have more normal radiosensitivity (D0 < 1.5 Gy; SCC-25,
SQ-38, and SQ-9G).
All the cell lines were aneuploid, having chromosome num
bers that ranged from about 56/cell to 82/cell. The DNA index
ranged from 1.81 to 2.57 (Table 1). There did not appear to be
any relation between radiosensitivity and either of these two
end points. Similarly, while these cell lines had different rates
of growth, neither faster nor slower growth rates were associ
ated with different radiation sensitivity (Table 1). Average gen
eration times ranged from 19.2 to 38.3 h. All the cell lines had
similar distributions of (i,, S, and G2-M cells in asynchronous
exponentially growing cultures (see below).
DNA double-strand break rejoining was measured in each
cell line after a 100-Gy exposure to 60Co -y-rays (Fig. 1). Some
of these data have been previously reported (17). By DNA
neutral elution analysis (17, 18), all cell lines were able to rejoin
approximately 90% of the double-strand breaks. Thus the ca
pacity for DNA double-strand break rejoining was not related
to radiation sensitivity. Instead, radiation sensitivity was a
function of how rapidly the DNA double-strand breaks were
rejoined. The radioresistant cell lines rejoined breaks faster
than the more sensitive cell lines. Rejoining of breaks was
essentially complete within 1 h after irradiation of JSQ-3, SCC12 B.2, and SQ-20B cells. In contrast, it took 2 or more h to
reach the same level of remaining damage (<10%) in the SCC25, SQ-38, and SQ-9G cell lines.
The induction and rejoining of chromosome breaks was
examined in each of the six cell lines after exposure in G, to 3Gy X-rays. The frequency of dicentric and ring aberrations as
a function of time between 1.5-Gy fractions was characteristic
5055
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RADIOSENSmVITY,
REPAIR, AND CHROMATIN
100
'75-k50-]5
40i[.W1-20100
\"ÉV
Ã-r-A-A^tr"1•^01234
50-
-A—
\0 D^-,^1\
^0— (l\J\J
'Ak\\\
25-n
25
.Br^•\
01234
01234TIME
(hours)
TIME (hours)
TIME (hours)
Fig. 2. Frequency of dicentric and ring chromosome aberrations per cell
induced in G, tumor cells as a function of time between two 1.5-Gy exposures.
A, radioresistant cell lines JSQ-3 (D), SCC-12 B.2 (A), and SQ-20B (O). B,
radiosensitive cell lines SCC-25 (A), SQ-38 (•),and SQ-9G (•).C, frequency of
dicentrics and rings per cell as a function of time between two 3.0-Gy exposures
in the radioresistant cell line SCC-12 B.2.
Table 2 Chromosome aberration induction in human tumor cell lines
lineJSQ-3SCC-12
Cell
counted100 deletions3
30
200200
550
deletions5
rings4
891557
350382
383
1903233
B.2SQ-20BSQ-38SCC-25SQ-9GDose(Gy)0
200100
30
500
30
100100
262
30
100200
1812721464Interstitial
6924
303Cells200200
200Terminal
STRUCTURE
rings (34.3 ±3.7) induced by X-rays in the radioresistant cell
lines was approximately one-half that found in the more radi
osensitive cell lines (136.7 ±27.3 and 67.0 ±4.0, respectively).
The cell cycle distributions measured in fixed whole cells
were similar for both radiosensitive and radioresistant cell lines,
as illustrated by the lines SCC-12 B.2 and SQ-9G (Fig. 3, A
and B). In contrast, the nucleoid fluorescence profile from the
radiosensitive cell lines exhibit a decreased resolution for all
cell cycle stages (see the SQ-9G example; Fig. 3, B and D), as
compared to radioresistant cell lines (see the SCC-12 B.2 ex
ample; Fig. 3, A and Q. These differences are documented for
all six cell lines by plotting a cumulative frequency distribution
for the Gìfluorescence peak. The radiosensitive lines all share
a decrease in maximum slope that is directly related to the
decreased resolution (Fig. 3, E and F).
The decreased resolution of the fluorescence histograms of
nucleoids from radiosensitive cell lines is produced by a varia
tion in the amount of ethidium bromide bound to each nucleoid.
Because nucleoids contain identical amounts of DNA as fixed
whole cells, the variation in ethidium bromide binding is most
likely due to the accessibility of DNA to the ethidium bromide
dye. Alterations in DNA supercoiling within the nucleoid struc
ture, for example, might affect the ability of ethidium bromide
to bind to all the DNA present.
DISCUSSION
21040132Dicentrics/
10857
119
for radioresistant and radiosensitive cells (Fig. 2). In the three
radioresistant cell lines, the frequency of dicentrics and rings
decreased when the two doses were split by l h and then changed
little as the time interval between fractions increased from 2 to
4 h. This result suggests that chromosome breaks remain open
for 1-2 h in the radioresistant cells. In contrast, for the more
radiosensitive cell lines (SCC-25, SQ-9G, and SQ-38), the
frequency of dicentric and ring aberrations remained elevated
when the doses were separated by l h and decreased as the time
interval between doses went from 2 to 3. Chromosome breaks
apparently remain open for 2-3 h in these cell lines. At this
dose of radiation (3 Gy), there were approximately twice as
many dicentrics and rings induced in the sensitive cell lines, as
compared to the resistant cell lines (see also Table 2). The
estimated restitution times and the kinetics of the split-dose
response were, however, independent of dose. In SCC-12 B.2
cells, doubling the overall dose to 6 Gy increased the aberration
frequency but did not change the estimate for chromosome
break restitution time or the shape of the response curve (Fig.
2Q.
The frequency of terminal deletions, dicentrics, rings, and
interstitial deletions induced in G, tumor cells by a 3-Gy
exposure to X-rays is shown in Table 2. In some cell lines,
there were relatively large numbers of aberrations in the nonirradiated samples. While there were some differences among
the cell lines in the frequency of radiation-induced terminal
deletions, the mean frequency/100 cells in radioresistant cell
lines (50.3 ±0.9) was approximately the same as in radiosen
sitive cell lines (47.3 ±8.2). In contrast, the frequency/100 cells
of both interstitial deletions (65.0 ±7.4) and dicentrics and
Human tumor cellular radiation sensitivity is a function of
the rate of DNA double-strand break rejoining. Radioresistant
cells rejoin radiation-induced DNA double-strand breaks in
about l h while more sensitive cell lines require 2 or more h to
rejoin the same fraction of damage (17) (Fig. 1). Measurement
of chromosome break-rejoining rates yield similar conclusions.
Radioresistant cell lines rejoin chromosome breaks in about 1
h of irradiation, while the more sensitive cell lines require 2-4
h to complete repair (Fig. 2). These results suggest that the
0
20
40
60
80
IOO 120 140
Fluorescence Intensity
Fig. 3. Cell cycle and nucleoid analysis. / and B, fluorescence histogram
showing distribution of cells within the cell cycle in exponentially growing cultures
of SCC-12 B.2 (A) and SQ-9G (B) cells. Cand D, fluorescence histogram showing
distribution of ethidium bromide-stained nucleoids from exponentially growing
cultures of SCC-12 B.2 (Q and SQ-9G (D) cells. E and F, cumulative frequency
histogam of the (., fluorescence peak in ethidium bromide-stained nucleoids for
(in £)radioresistant cell lines JSQ-3 (A), SCC-12 B.2 (O), and SQ-20B (D) and
(in f) radiosensitivity cell lines SCC-25 (O), SQ-38 (D), and SQ-9G (A).
5056
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RADIOSENSITIVITY. REPAIR, AND CHROMATIN STRUCTURE
DNA damage measured by DNA neutral elution is a marker
for chromosome breaks as well.
In the DNA elution studies, all the cell lines rejoined approx
imately the same fraction of DNA double-strand breaks. There
fore there should be no difference between sensitive and resist
ant cell lines in the frequency of unrejoined chromosome breaks.
This is what is noted. The frequency of terminal deletions is
essentially the same in resistant and sensitive cell lines (Table
2). However, in cells where DNA/chromosome breaks remain
open longer, as chromatin structure changes with time after
irradiation the probability of a chromosome exchange (misrepair event) might be greater than in a cell where the breaks are
more rapidly sealed. As seen in Table 2, radiosensitive cell lines
have twice the frequency of induced chromosome exchangetype aberrations as do resistant cell lines. These results suggest
that faster rates of DNA/chromosome break rejoining are as
sociated with greater radioresistance because they result in
fewer lethal misrepair events.
The basis for different rates of rejoining of DNA and chro
mosome breaks is not known. The different rates of rejoining
are not a function of DNA content or chromosome number.
They also appear to be unrelated to average generation time or
the distribution of cells within the cell cycle. There were quali
tative differences in the fluorescence patterns of nucleoids de
rived from radiosensitive and radioresistant cell lines, suggest
ing that radiation responses might be related to chromatin
structure. It is possible that the chromatin structure in radi
oresistant cell lines is such that radiation-induced breaks are
held in an appropriate configuration for a rapid and accurate
rejoining. In contrast, in radiosensitive cell lines, breaks might
not be held in such a favorable configuration. Alterations in
DNA organization within radiosensitive cells may favor misrepair events (chromosome exchange-type aberrations). This
would explain why, in radiosensitive cell lines, the frequency of
dicentrics and rings seen after two 1.5-Gy X-ray doses split by
l h is actually the same or higher than that seen when the two
doses are given at the same time.
These results with human tumor cell lines are quite different
from those reported for rodent cell lines. For most studies on
the basis for radiation sensitivity in mutant mouse or hamster
cell lines, radiation sensitivity is found to be associated with a
reduced capacity to rejoin DNA double-strand breaks (5-7). In
contrast, radiation sensitivity in human tumor cell lines is
associated with a slower rate of DNA/chromosome break re
joining. These differences could reflect inherent differences
between human and rodent cells. Alternatively, these differences
could be due to differences between tumor and nontumor cell
lines. Further study of these human tumor cell lines should
provide more information on human cellular radiation re
sponses as well as the involvement of chromatin structure in
repair and chromosome aberration induction. Furthermore,
because it has been suggested that inherent radioresistance in
human tumor cells might be an important factor in the failure
of radiation to sterilize certain tumors (11-13), these studies
might suggest predictive assays of tumor response as well as
alternative approaches to standard radiation therapy in resistant
tumors.
ACKNOWLEDGMENTS
The authors would like to acknowledge the technical assistance of
M. A. Beckett, R. Mustafi, J. M. Perrin, and S. M. Giovanazzi and the
advice of Drs. R. R. Weichselbaum and D. J. Grdina.
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Association among DNA/Chromosome Break Rejoining Rates,
Chromatin Structure Alterations, and Radiation Sensitivity in
Human Cell Lines
Jeffrey L. Schwartz and Andrew T. M. Vaughan
Cancer Res 1989;49:5054-5057.
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