(CANCER RESEARCH 50, 6575-6579. October 15. 1990]
Induction and Repair of DNA Double Strand Breaks in Radiation-resistant Cells
Obtained by Transformation of Primary Rat Embryo Cells with the Oncogenes
H-ras and v-mycl
George Iliakis,2 Lorenz Metzger, Ruth J. Muschel, and W. Gillies McKenna
Department of Radiation Oncology and Nuclear Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 fO. /., L. M.J, and Departments of Pathology
and Laboratory Medicine ¡R.J. M.J and of Radiation Oncology [W. G. M.J, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
ABSTRACT
Rat embryo cells (REC) transformed by the H-ras oncogene plus the
cooperating oncogene »-m.trare highly resistant to ionizing radiation as
compared with the nontransformed parent cells, REC, or immortalized
REC. In an attempt to understand the potential mechanism of resistance
in these cells, the induction and repair of double strand breaks (dsb) in
DNA were measured in a ll-ras plus \-myc transformed (3.7) and an
immortalized REC (mycREC) line using pulsed field gel electrophoresis.
Cells were irradiated in the exponential phase of growth, and the amount
of DNA dsb present was quantified by measuring the fraction of DNA
activity released from the agarose plugs in which cells were embedded.
Similar values of the fraction of DNA activity released were measured
for both cell lines at equal X-ray doses, after correction for differences in
cell cycle distribution, suggesting a similar induction of DNA dsb per Gy.
Repair of DNA dsb measured after exposure to 40 Gy of X-rays was
similar in both cell lines and displayed a fast and a slow component. The
fast component had a 50% repair time of approximately 12 min, and the
slow component, 50% repair time of about 3 h. These results suggest
that the relative radioresistance of 3.7 cells is not conferred by a decrease
in the amount of DNA dsb induced per Gy per dalton or by alterations
in the capacity of the cells to repair DNA dsb. It is hypothesized that
alterations in the expression of potentially lethal damage underlie this
phenomenon.
INTRODUCTION
Evidence accumulates that expression of certain types of
oncogenes in cells of various origins can confer resistance to
ionizing radiation. Thus, Chang et al. (1) reported that normal
fibroblasts from skin biopsies of patients with the Li-Fraumeni
syndrome showed overexpression of the myc and activation of
the raf oncogene and displayed at the same time increased
resistance to ionizing radiation. Furthermore, Kasid et al. (2)
demonstrated that a cell line developed from a clinically radioresistant human squamous cell carcinoma was also character
ized by an activation of the raf oncogene. FitzGerald et al. (3)
observed increased radioresistance in NIH3T3 cells containing
the human N-ras gene. Increased radioresistance in NIH3T3
cells transfected with various forms of the ras oncogene has
also been observed by Sklar (4). More recently, evidence has
been presented that the effect of oncogenes such as H-ras in
conferring radioresistance can be augmented in REC3 if cotransfected with the oncogene myc (5, 6). These findings correlate
genetic alterations with modifications in the intrinsic radiosenReceived 5/4/90; accepted 7/13/90.
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 National Cancer Institute Grants CA 45557 and
CA 42026 awarded from NIH, Department of Health and Human Services.
Presented in part at the 38th Annual Meeting of the Radiation Research Society,
held in New Orleans. LA, on April 7-12. 1990.
2To whom requests for reprints should be addressed.
3The abbreviations used are: REC, rat embryo cells; dsb, double strand
break(s); AFIGE, asymmetric field inversion gel electrophoresis; CHO, Chinese
hamster ovary; FAR, fraction of activity released; t50. time required to complete
50% of the repair.
sitivity of a cell and may have implications for the treatment of
human tumors by ionizing radiation. The effect of the activated
ras oncogene on radiation resistance may be of great clinical
significance, since 90% of human pancreatic carcinomas (7),
50% of colon carcinomas (8, 9), and 20% of lung cancers (10)
have oncogenic mutations in ras genes. The myc oncogene
family is often amplified or overexpressed in a wide variety of
tumors (11). It is possible that the presence of certain types of
oncogenes in human tumors reduces their radiosensitivity, thus
inadvertently affecting the outcome of treatment by radiation.
Although radioresistance mediated by oncogenic transfor
mation has been demonstrated in a number of cell lines and the
associated complications for the treatment by radiation of hu
man tumors discussed (see previous paragraph for references),
little is known about the mechanism that underlies the phenom
enon. Yet elucidation of the mechanism underlying reduced
radiosensitivity in cell lines transfected with certain families of
oncogenes may help devise strategies for overcoming problems
in the radiotherapy of malignancies that happen to express
these genes.
There is compelling evidence suggesting that the target of
ionizing radiation-induced cell killing is the DNA (12), and
that DNA double strand breaks are the lethal lesions. For
example, a direct correlation between unrepaired DNA dsb and
cell killing was established in DNA dsb repair-deficient mutants
of yeast (13-15), and radiosensitive mutant cell lines have been
isolated that are partially defective in their ability to repair
these lesions (16-19). It was, therefore, inquired whether the
reduction in radiosensitivity observed in ras-transfected cell
lines is associated with modulations in either the induction or
the repair of DNA dsb.
A radiation-resistant cell line obtained by cotransfection of
REC with the oncogenes H-ras and \-myc (3.7) was used for
the experiments (6). The results were compared to those ob
tained with a cell line immortalized by transfection of REC
with c-myc (mycREC) that displayed normal sensitivity to
ionizing radiation exposure (6). Induction and repair of DNA
dsb were assayed by a recently developed pulsed field gel elec
trophoresis technique, the AFIGE (20, 21). Pulsed field gel
electrophoresis, originally devised for the separation of DNA
molecules up to several million base pairs (22-26), has recently
been shown to be a powerful assay for the measurement of
induction and repair of DNA dsb in mammalian cells. Thus,
clamped homogeneous electric field gel electrophoresis (27),
transverse alternating field electrophoresis (28-30), and AFIGE
(20,21, 31, 32) have been successfully used for the measurement
of dsb in the DNA of mammalian cells.
Here, we report experiments designed to study induction and
repair of DNA dsb in irradiated 3.7 and mycREC cells, using
AFIGE. The results obtained indicate similar induction and
similar rates of repair for DNA dsb in both cell lines and
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DNA dsb REPAIR IN ONCOGENE-TRANSFORMED
suggest that the mechanism that confers radiation resistance in
3.7 does not rely on modulation of these parameters.
MATERIALS AND METHODS
Cell Culture. Cell lines 3.7 and mycREC derived from primary rat
embryo fibroblasts as previously described (6), were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated
fetal bovine serum, at 37°Cin a humidified incubator in an atmosphere
of 5% CO2 and 95% air. Cells were kept in a quasicontinuous culture
by subculturing every second day at an initial density of 5 to 10 x IO5
cells/flask (80 cm2 tissue culture flask, 20 ml of medium). For experi
ments with 3.7 cells, 1 to 3 x IO6 cells were seeded in 80 cm2 tissue
culture flasks (20 ml of medium) and were used 2 days later (cells were
still in the exponential phase of growth); different numbers of cells
were seeded per flask, within the above given range, in order to obtain
cultures with different distributions of cells throughout the cycle (see
"Results"). In general, high initial cell numbers per flask resulted in
switching unit and a custom-made device with two rheostats to reduce
the voltage in forward direction. After the end of the electrophoresis
time, gels were photographed under UV and subsequently cut to sepa
rate the plug from the lane for each sample. The 14Cactivity of each
piece was measured in a scintillation counter and the FAR was calcu
lated as lane DPM divided by total (plug + lane) DPM. The FAR in
cells that were not exposed to radiation (termed background) was
determined in each experiment (1 to 3%) and was subtracted from the
FAR values obtained in irradiated cells before plotting. There was no
change in the FAR of unirradiated cells kept in suspension for repair
up to 4 h.
Biphasic repair kinetics was observed throughout this work, and
repair time constants for the fast (a) and the low (b) component were
calculated by fitting the results obtained to the equation
FAR = A x 10-"' + B x ¡Or"
using a two-step procedure (A and B represent the fraction of DNA dsb
repaired with fast and slow kinetics, respectively). In the first step, data
points between 60 and 240 min were used to determine B and b by
fitting to the equation FAR = B x 10"*'. In the second step, A and a
cell populations enriched in d cells after 2 days of growth. MR cells
were seeded at 5 x 10' cells per 150 cm2 dish (20 ml of medium) and
were used after 2 days of growth. Cells were labeled with 0.74 kBq/ml
of [l'1C]thymidine plus 5 ^M thymidine, added at the time of culture
preparation. Distribution of cells throughout the cell cycle was meas
ured in each experiment using a mercury arc lamp flow cytometer
(Partee, PAS II). For this purpose, cells were stained by direct suspen
sion (about 5 x 10' cells) in a solution (1 ml) containing 0.1 M Tris,
0.1 M NaCl, 5 mM MgCl2, 0.05% Triton X-100, and 2 Mg/ml of 4',6diamidino-2-phenyl-indole (DAPI). Data were collected in a computer
equipped with the necessary software to calculate the fraction of cells
in d, S, and G2 + M phase. The cell cycle distributions thus obtained
were used for the interpretation of the results at the DNA level.
Compared with previously published data (6), small differences in the
distribution of cells through the cycle were observed. These differences
are probably due to slight differences in the growth protocols or the
serum lot used; however, alterations in the growth characteristics of the
cells cannot be excluded. The clonogenic assay was used to measure
cell survival after exposure to various doses of X-rays; the parameters
of the resulting survival curves were derived as described previously (6).
Measurement of Induction and Repair of DNA dsb. Dose-response
curves for the induction of DNA dsb were measured using a pulsed
Held gel electrophoresis technique, the AFIGE (20, 21, 31, 32). For
this purpose cells were trypsinized, spun down, and suspended in 1%
agarose at a concentration of 3 x 106/ml (FMC InCert) at 37°C.This
suspension was pipetted into glass tubes, was allowed to cool in ice
until solidification, and was then cut into 3x5 mm pieces containing
about 1 x IO5 cells. Cells were irradiated on ice, using a therapeutic
Siemens X-ray machine operated at 250 kV, 15 mA with a 2 mm Al
filter. Immediately after irradiation cells were lysed for 20 h at 50°Cin
a solution containing 0.5 M EDTA, 0.01 M Tris, 2% N-lauryl sarcosine
(NLS), and 0. l mg/ml of proteinase K at pH 8.O. Subsequently, samples
were washed for l h in 0.1 MEDTA, 0.01 M Tris, pH 8.0, and were
then treated for l h at 37"C with 0.1 mg/ml of RNase A (boiled 10
CELLS
were determined by fitting the data between 0 and 30 min to the
equation FAR - B x 10"*' = A x 10~". The findings presented here
have been reproduced in 3 to 7 independent experiments,
results are shown as the resulting mean ±standard error.
and the
RESULTS
In Fig. 1, recent survival curves are shown that were obtained
with exponentially growing mycREC (A) and 3.7 (•)cells. The
data demonstrate a marked resistance to ionizing radiation
exposure for 3.7 cells and confirm previously published obser
vations (6).
In Fig. 2, dose-response curves are shown for the induction
of damage in the DNA, as measured by AFIGE in the range
between 0 and 70 Gy. Plotted in the figure is the FAR, measured
as described under "Materials and Methods," as a function of
the radiation dose for 3.7 and mycREC cells grown under
various conditions. Fig. 2, A, depicts results obtained with
exponentially growing mycRec cells. In the range of doses
examined, the results obtained could be adequately fitted by a
straight line. It has been pointed out elsewhere (31, 32), how-
min to eliminate DNase contamination) dissolved in the same solution.
To measure repair of DNA dsb, cells were suspended in fresh growth
medium, irradiated with 40 Gy, and held in suspension in glass tubes
at 37°Cplus 5% CO2 to allow repair (6 x IO6cells/ml). Aliquots were
taken after various times, and cells were mixed quickly with melted
agarose (37°C,some repair is possible during this step) and cooled by
submersion in ice. Subsequently, plugs were made, and cells were lysed
as described above. Results obtained with CHO cells suggested that
incubation of cells in suspension at this concentration did not affect
their ability to repair as compared with cells kept in monolayer.
Asymmetric Field Inversion Gel Electrophoresis. Electrophoresis was
carried out for 40 h in half-strength 45 miviTris, 45 m\i boric acid, 1.5
mM EDTA, pH 8.2, at 10°C,in BRL (H4) horizontal boxes, in 0.5%
agarose gels (FMC SeaKem GTG) cast in the presence of 0.5 Mg/ml of
ri Indium bromide. AFIGE was performed by applying cycles of 1.25
V/cm for 900 s in the direction of net DNA migration, and 5 V/cm for
75 s in reverse direction, using a power supply, an IBI (Minipulse)
10
Dose, Gy
Fig. 1. Clonogenic radiation survival curves for mycREC (A, £>„
= 1.1 Gy)
and 3.7 (•,£>0
= 2.2 Gy) cells. Fitting of the data to a linear quadratic equation
gave the following results: MycRec cells: a = 0.16 Gy"1, ß
= 0.74 Gy~:; 3.7 cells:
a = 0.0 Gy-1, 0 = 0.59 Gy"2.
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DNA dsb REPAIR IN ONCOGENE-TRANSFORMED
slightly higher induction of damage (DNA dsb) in 3.7 cells, the
effect is small and cannot be confirmed without experiments
with synchronized cell populations. Within the uncertainties of
this type of experiments, these results are consistent with sim
ilar induction of DNA dsb in mycREC and 3.7 cells.
In Fig. 3, repair kinetic values are shown for mycREC and
3.7 cells exposed to 40 Gy in the exponential phase of growth.
Plotted in the figure is the FAR as a function of the postirra
diation incubation time at 37"C. Cell populations similar to
0.7
MR (20-23% SI
0.6 -
• 3.7 (20-29% SI
O 3.7 (33-42% S)
0.5
"Ö 0.3
those used in the experiments described in Fig. 2 were used.
3.7 cells grown at high initial concentrations gave larger initial
(/ = 0) values for the FAR than did the corresponding mycREC
cells, in agreement with the results shown in Fig. 2. However,
the difference between mycREC and 3.7 cells grown after
seeding at an initial density of IO6 cells was smaller than that
•y
o.oo0
CELLS
50
60
70
Dose/Gy
Fig. 2. Dose-response curves for the FAR (reflecting induction of DNA dsb),
as measured by AFIGE, in exponentially growing mycREC (MR) (A) and 3.7 (•
and O) cells. Experiments carried out with 3.7 cell cultures containing different
amounts of S-phase cells are shown (O and •).Points, mean; öars,SD.
ever, that this way of fitting is only a first approximation that
is valid in the low-dose region; it does not assume or imply a
linear dependence of the FAR on dose in the entire range of
doses where measurements are possible. The results obtained
with 3.7 cells seeded at an initial density of approximately IO6
cells per flask are shown in Fig. 2, O. Once again, the results
obtained could be fitted by a straight line. For a given dose of
radiation, the FAR in 3.7 cells was slightly lower than that of
mycREC cells. This difference could, in principle, be attributed
to differences between the two cell lines in the induction of
DNA dsb per Gy per dalton. However, experiments performed
with synchronized populations of CHO cells (31, 32) suggest
that such conclusions are only valid when the distribution of
cells throughout the cycle in the two cell lines is identical. Flow
cytometry, carried out in parallel to the experiments shown in
Fig. 2, indicated that MR cells had a lower percentage of Sphase cells (20 to 23%) than 3.7 cells when seeded at approxi
mately IO6cells/dish (33 to 42%).
Since it has previously been shown that S-phase cells show a
lower FAR per Gy than cells irradiated in Gìor G2, for the
same induction of DNA dsb per Gy per dalton (31, 32), exper
iments were carried out with 3.7 cells grown under conditions
that reduced the percentage of S-phase cells in the population.
Reduction in the fraction of S-phase cells in 3.7 cells was
effected by preparing cultures at higher initial cell concentra
tions (1.5 to 3 x IO6 cells/dish) and allowing cells to grow for
2 days. Under these conditions cell populations were obtained
with a fraction of S-phase cells (20 to 29%) closer to that
measured in mycREC cells. Fig. 2, •,depicts results obtained
for DNA damage induction in these cultures. The FAR for a
given dose of radiation was slightly higher than that measured
in mycREC cells. Taken together, the set of results presented
with 3.7 and mycREC cells in Fig. 2 suggests that only small
differences exist in the DNA damage induction dose-response
curves which can be attributed to changes in the FAR per Gy
caused by differences in the distribution of cells through the
cycle. Although the available results appear consistent with a
shown in Fig. 2, probably due to experimental variation. De
spite the small differences in the initial levels, the FAR rapidly
decreased as a function of time according to biphasic kinetics.
The reduction in the FAR with increasing postirradiation in
cubation time is caused by a restoration of the molecular size
of the DNA to values closer to those obtained with nonirradiated cells and reflects repair of DNA dsb (21, 27).
To facilitate comparison of the repair kinetics measured in
the different cell lines used after growth under various condi
tions, the FAR values of Fig. 3 were normalized to the values
of FAR measured at t = 0, and the results obtained are shown
in Fig. 4. To prevent congestion in the graph and to facilitate
comparison of the different sets of data, a 1-h shift was intro
duced for each set during plotting. The results indicate biphasic
repair kinetics for radiation-induced DNA dsb for both cell
lines. Regression analysis was carried out using these data,
assuming two exponential functions, in order to calculate the
tso values for the slow and the fast repair components, as well
as the fraction of DNA dsb repaired with slow kinetics. The
results obtained from this analysis are summarized in Table 1.
No significant differences were observed in the repair time
constants or in the fraction of DNA dsb repaired with slow
kinetics between mycREC and 3.7 cells. The fast component
180
210
240
Time/min
Fig. 3. Repair of exponentially growing mycREC (MR) and 3.7 cells after
exposure to 40 Gy of X-rays. Plotted is the fraction of activity released from the
plug as a function of the repair time. Other details as in Fig. 2.
6577
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DNA dsb REPAIR IN ONCOCENE-TRANSFORMED
were not due to differences in the induction of DNA dsb per
Gy per dalton, but rather to fluctuations in the sensitivity of
the assay, caused by alterations in the electrophoretic properties
of the DNA molecules during S phase. It was proposed that
partly replicated DNA molecules display reduced mobility due
to the presence of replication forks and eyes. It is interesting
that similar processes were also suggested by results obtained
using the nonunwinding filter elution (33). Since the kinetics
of DNA dsb repair was also similar in mycREC and 3.7 cells,
it can also be hypothesized that the increased radioresistance
of the latter cells does not derive from genetic changes that
modify repair kinetics.
Although the results at the DNA level did not unveil differ
ences in response sufficient to explain the observed differences
in radiosensitivity between mycREC and 3.7 cells, examination
of other endpoints associated with the radiation response of
these cells did yield substantial differences. Thus, irradiated 3.7
cells exhibited a significantly longer G2 delay per Gy than did
mycREC cells (34) and showed an increased sensitivity to
radiation-induced inhibition of DNA replication.4 These results
§.
£
* MI;
• 3.7 (20-29% S)
o
3.7 (33-42% S)
0 30 60 90 120150180210240270300330360
Time/min
Fig. 4. Results from Fig. 3 normalized to the FAR measured at ( = 0. To
facilitate comparison, each set of data was shifted by 1 h. Other details as in Fig.
3.
Table 1 Repair time constants calculated from Fig. 3, assuming biphasic
exponential repair kinetics
CellsmycREC3.7
CELLS
of
damage
repaired by
of slow
slow
soof fast
componenl/min11.8(11.4-12.3)°
component/min188(172-208)
component0.23
173(157-193)
(20-29% S phase) 11.6(10.6-12.6)
0.35
189(119-454)Fraction 0.23
3.7 (33-42% S phase)t 11.2(10.5-12.1)tso
' Numbers in parentheses. 68% significance intervals of the repair time con
stant determination.
are the mirror image of those obtained with ataxia telangiectasia
cells (35) and may suggest mechanisms by which increased
resistance to radiation is conferred in 3.7 cells. Of importance
also is the observation that the difference in radiosensitivity
between mycREC and 3.7 cells is significantly reduced by
postirradiation incubation with caffeine,5 that is known to sen
sitize cells to radiation by fixing potentially lethal damage (3639).
In summary, the results presented here indicate that the
increased resistance to ionizing radiation of 3.7 cells may not
be due to modulations in the induction and repair of DNA dsb.
Reduction in potentially lethal damage fixation, mediated by
alterations in the delays induced by radiation in the progression
of cells through the cycle, probably underlies the observed
differences in radiosensitivity between mycREC and 3.7 cells.
had tso values of about 12 min, whereas the slow component
had tio values of about 3 h for both cell lines, and 25 to 35% of
the DNA dsb were repaired with slow kinetics in every case
examined.
ACKNOWLEDGMENTS
DISCUSSION
REFERENCES
Special thanks go to Giridhar Vedala and Mary Jackson for excellent
technical assistance and to Nancy Mott for secretarial help.
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6579
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1990 American Association for Cancer Research.
Induction and Repair of DNA Double Strand Breaks in
Radiation-resistant Cells Obtained by Transformation of Primary
Rat Embryo Cells with the Oncogenes H- ras and v-myc
George Iliakis, Lorenz Metzger, Ruth J. Muschel, et al.
Cancer Res 1990;50:6575-6579.
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