[CANCER RESEARCH 39, 929-933, March 1979]
0008-5472/79/0039-0000$02.00
Increased Susceptibility of Mouse Cells to Fluorescent Light-induced
Chromosome Damage after Long-Term Culture and Malignant
Transformation
Ram Parshad, Katherine K. Sanford,1 Robert E. Tarone, Gary M. Jones, and Anne E. Baeck
Department
of Pathology,HowardUniversityCollegeof Medicine,Washington,
D. C.20059(A. P.),andLaboratory
of CellularandMolecularBiology(K.K.
S., G. M. J., A. E. B.) and Biometry Branch fR. E. T.j, National Cancer Institute, NIH, Bethesda, Maryland 20014
ABSTRACT
Exposure of mouse cells in culture to fluorescent light
has been shown to produce chromatid breaks and ex
changes. Hydrogen peroxide formed in the cell during
illumination has been implicated as the causative agent.
The present results indicate that susceptibility to light
induced chromosome damage increases with time in cul
ture and seems to be associated with or requisite for the
spontaneous malignant transformation of mouse cells. All
three cell lines followed during long-term culture that either
became tumorigenic or showed cytological evidence of
neoplastic transformation developed a concomitant in
crease in susceptibility. In three additional cell lines, sus
ceptibility to light-induced chromatid damage was signifi
cantly increased in the spontaneously transformed malig
nant cells as compared with their nonneoplastic precursors.
The increased susceptibility is not simply the result of long
term culture, since three other nonneoplastic cell lines after
prolonged culture were significantly less susceptible than
their malignant counterparts. Increased susceptibility to
light-induced chromatid damage could result from impaired
DNA repair or from the loss of defense mechanisms for
destroying H@O2
or scavenging free radicals.
INTRODUCTION
Previousstudies (6, 11, 12, 16) haveshown that exposure
of mouse cell cultures to fluorescent light produces chro
matid breaks and exchanges and DNA cross-linkage. The
chromosome damage was observed in cultures exposed to
room fluorescent lights during routine handling, as well as
after more prolonged exposure (20 hr) at 37°to cool-white
fluorescent light with an intensity of —iSOfootcandles. The
effective wavelength for the chromatid breaks and ex
changes was found to be 405 nm in the visible mange(i3).
We infer that H@O2produced in the cells is the causative
agent because the light-induced chromosome damage can
be minimized by lowering the oxygen concentration in the
gas phase from atmospheric (i8%) to 0 or 1%, can be
preventedto a greatdegree by adding glutathione
and
ascorbic acid or the enzyme catalase (hydrogen peroxide:
hydrogen peroxide oxidoreductase, EC i.ii.i.6)
to the
medium, and can occur inlight-exposed
cellswashed and
suspended in phosphate-buffered 0.85% NaCI solution.
During the course of these experiments, we observed that
the susceptibility of mouse fibroblasts to light-induced
chromosome damage seemed to increase with serial pas
sage and time in culture. The present results confirm this
impression and further suggest that the increased suscep
tibility may be associated with or requisite for “sponta
neous―malignant transformation in long-term lines of
mouse cells.
MATERIALSAND METHODS
Source of Cells. All cell lines used in this study originated
from 10- to 13-day C3H/HeN or C3Hf/HeN mouse embryos
except the derivatives of NCTC 8466, which originated from
the lung tissue of four 59-day-old male C57BL/6N mice (i 1,
17). The early history and properties of these lines (except
NCTC 9255 and 9258) have been described (3, 7, i 4-i 6).
With the exception
of NCTC lines 9255, 9258, and 916S,
cells were derived from frozen stocks. Cultures were carried
in Pyrex T-i 5 flasks in 3 ml of either Dy2 or NCTC 135 with
iO% FBS; medium
was renewed
3 times
weekly
when
cultures were gassed with a humidified mixture of 10% CO2
in air (18% 02) or iO% C02:0% 02:90% N@as indicated. No
antibiotics were used except for the derivatives from lung
tissue, which received gentamicin, 0.i mg/mI (Scheming
Corp., Kenilworth, N. J.), but antibiotics were not used
during experimental treatment, and these cells as well as
the other cell lines tested were negative for Mycoplasma or
microbial contamination (2). Cells were subcultumed when
confluent by a brief rinse with Versene (1:5000; Micmobio
logical Associates, Bethesda, Md.) and subsequent dispem
sion with Vemsene:trypsin Mixture ATV (8). Cell lines me
ferred to as malignant had been assayed in syngeneic mice
and found to grow as invasive, transplantable sarcomas (3,
7, i4—i6).
Conditions for Light Exposure. Stock and control cul
tures and culture medium were never exposed to light of a
wavelength below 500 nm, as they were handled under gold
or red fluorescent lights. To examine the effect of light,
cells
were inoculated
at0.5to 1 x 10@cehls/2
ml ofculture
medium into Leighton tubes, each containing a 9- x SO-mm
coverslip (No. 1 thickness; Bellco Glass Co., Vinehand, N.
J.), and tubes were gassed with iO% CO2 in air and stop
pemed.After 24 hr incubation at 37°,the experimental cells
were exposed through the Pyrex Leighton tube for 20 hr at
370
to
a
desk
lamp
fitted
with
2
cool-white
Westinghouse
bulbs (F15T8-CW). The lamp was at a distance of 40.6 cm
I To
whom
requests
for
reprints
should
be
addressed,
at
In
Vitro
Carci
nogenesis Section, National Cancer Institute, NIH, Building 37, Room 2D02.
Bethesda. Md. 20014.
Received September 21, 1978; accepted December 6, 1978.
2 The
abbreviations
used
are:
Dv,
Dulbecco-Vogt
medium;
FBS,
fetal
bovine serum.
MARCH 1979
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929
R. Parshad et a!.
and supplied iSO footcandles (4.6 watts/sq m) at the level
of the growth surface as measured by a Weston Model 6i4
light meter.
Chromosome Analysis. Immediately after light exposure,
0.1 @gof Colcemid (Grand Island Biological Co. , Grand
Island, N. Y.) reconstituted in phosphate-buffered saline (1
mM KH2PO4:S.6 mM Na2HPO4, pH 7.4) per ml of culture
medium was added to 4 control and 4 experimental cul
tures, which were incubated at 37°for 1.5 hr to produce
mitotic arrest. Cells were treated with hypotonic solution
and fixed in situ; coverships were air-dried, stained, and
mounted as described previously (6). In each experiment,
approximately 200 intact metaphase plates were randomly
selected from coded experimental and control cultures for
analyses. Except as indicated, the x2 test of heterogeneity
was applied for statistical evaluation of the data. This test
takes into account both the number of cells with each
chromosome defect and the number of such defects in
each cell.
RESULTS
Influence of Period in Culture on Light Susceptibility.
Table i summarizes the results of a series of tests on 2
sublines of NCTC 8466 grown in DV with 10% FBS. Cells
were light exposed after various periods and passages in
culture; some ofthe data for subhine NCTC 9i86 have been
reported previously as control values (12). The light-ax
posed cells showed a significant increase in chromatid
breaks in all tests and a significant increase in chromatid
exchanges in many tests. There was no significant change
in the frequency of minute or metacentric chromosomes in
exposed cells compared to shielded cells; however, the
frequency of metacentric chromosomes in both shielded
and exposed cells increased with time in culture. Linear
Chromosome
abnormalities
in sublines
regression analysis using the mean number of metacentric
chromosomes per cell as the dependent variable and the
passage number as the independent variable yielded
regression coefficients of 0.060 for shielded 9186 cells (p
=
0.001),
0.053
for
exposed
9186
cells
(p
=
0.003),
0.018
for shielded 9266 cells (p = 0.042), and 0.019 for exposed
9266 cells (p = 0.045). Similarly, the frequency of minutes
in subline 9266 increased with time in culture, yielding
regression coefficients of 0.028 for shielded cells (p <
0.001 ) and 0.032 for exposed cells (p < 0.001 ). This in
crease occurred even though the stock cultures were not
exposed to light of a wavelength <500 nm and were also
flushed with 0% 02 at each fluid renewal.
An important finding was an increase in susceptibility to
light-induced chromatid breaks and exchanges occurring
after about 400 days in vitro. A comparison of exposed cells
tested before and after 400 days in culture revealed that the
exposed cells tested after 400 days had significantly greater
mean numbers of chromatid breaks (p < 10@) and chro
matid exchanges (p < 0.001) per cell. When cells of this
line were assayed in syngeneic hosts at 413 days, they grew
as sarcomas. These observations raised questions as to
whether mouse fibroblasts generally show an increased
susceptibility to light-induced chromosome damage with
time in culture and whether there is any relationship be
tween this increased susceptibility and spontaneous mahig
nant transformation.
Table 2 summarizes the results of light exposure on 3 cell
lines grown in Medium NCTC i 35 with 10% FBS and tested
after
2 different
periods
in culture.
Certain
lines were
carried as 2 subhines with 0 or 18% 02 in the gas phase as
indicated. In the 3 sublines of NCTC 9258 and 9165, the
frequency of light-induced chromatid breaks/cell increased
significantly after prolonged culture (p < 0.007). The 2
sublines of NCTC 9255 examined relatively early in culture
Table 1
of NCTC 8466 mouse cells shielded and light exposed
periods of culture in DV with 10% FBS
for 20 hr after various
no./ceIIC@:aChrornatidex
NCTCsublineTime
in vitro (days)Av.
MinutesbMetacentricsSCESES
E9186167(10)d
ES
0.105
0.041
189 (12)
0.025
0.285
0
o.o5oe
0.015
0.045
0.020
0.065
231 (16)
0.025
0.405
245(18)
0.015
0.210
273(22)
0.030
0.285
0
0
0
0.O40@
0.025
0.040@'
0.015
0.010
0.030
0.025
0.015
0.010
0.125
0.175
0.600
0.095
0.130
0.550
301 (26)
0.015
0.310
0
0.025
0.010
0.010
1 .055
1.015
303(26)
310(27)
401 (39)
0.020
0.040
0.040
0.087
0.260
0.300
0.700
0.733
0.010
0.013
0
0.007
0.060
0.040
0.175e
0.147e
0.8259266302(26)
329 (29)0.044
0.935
0.0350.006
0.2400.011
00.10Sf' 0.0250.0270.025
0.0050.279
0.030
0.060
0.060
0.355
0.480
464(46)
0.960
1.0300 00.070@ 0.105e0.030
541(57)0.040 0.0300.350
(1
In
all
tests,
exposed
cells
have
a
significantly
increased
mean
number
of
chrornatid
e
930
Exposed
cells
have
a significantly
increased
mean
number
of
chromatid
exchanges
(p
<
0.0001).
of the mouse
(p
0.010
0.020
0.080
0.255
0.771
0.460 0.465
1.1200.060
breaks
b ‘Minutes―
are chromosomes
less than one-half the length of the shortest chromosome
C @,shielded;
E, light exposed.
d Numbers
in parentheses, passage number.
0.030
0.093
0.245
0.720
0.060
0.093
0.390
0.520
<
karyotype.
0.05).
CANCER RESEARCH VOL. 39
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Susceptibility to Chromosome Damage
when still nonneoplastic
days in culture (p < 0.0001). At this time, the cells were
highly susceptible to light-induced chromatid breaks and
exchanges and were cytologically diagnosed to be malig
showed no increase in susceptibil
ity. Further, a combined analysis of all sublines revealed a
highly
significant
increase
after prolonged
culture
in the
proportion of light-exposed cells with chromatid breaks (p
nant by 250 days in culture.
<
with 18% 02 in the gas phase also showed an increased
0.0001).
The
combined
analyses
of
all subhines
also
Cells of subhine 9258 B grown
showed that the exposed cells had a significant increase in
susceptibility
chromatid
changes by 206 days in culture and became malignant
exchanges
associated
with time in culture
(p =
to light-induced
chromatid
breaks
and ax
0.003).
between 155 and 304 days in culture.
In shielded cells, no significant increase in frequency of
chromatid
breaks or exchanges
occurred
with time in
culture except for an increase in breaks in line 9165 by 271
Susceptibility to Light-induced Chromatid Damage and
Malignant Transformation in Culture. To evaluate further
the possible relationship between susceptibility to light
induced chromatid damage and malignant transformation,
Table 2
Chromatid
breaks and exchanges
3 lines of mouse cells that had been cryopreserved
in mouse embryo cell lines
shielded and light-exposed after various periods of culture in
NCTC 135 with 10% FBS
@
@___-@__@_-_____-_@---;i
@----@ -@-@-@-
Table
Av. no/cell
NCTC
cell line
%
02
Time in
vitro
Chromatid breaks
(days)
b
@
9255A
0
9255B
18
99(15)'
127(19)
3
compares
exchanges
Chromatid exchanges
the
in these
frequency
lines
the
E
other
hand,
light
0.007
0
0.013
(p = 0.002
0.012
0
0.024
cantly
0.025
0.027
0.005
0.007
0.005
0.007
and exchanges after
values, seeTable3).
99(15)
0.020
0.026
of
chmomatid exchanges
breaks
before
and
and
after
trans
to light induced
a sign if i
(p = O.OOi to <10 6). On
induced
a
significant
increase
in
only after the cells became malignant
to <1O6).
increased
chromatid
examined
formation.
In all assays, exposure
cant increase
in chromatid
breaks
S
0.013
0
127(19)
before
and after spontaneous malignant transformation were
thawed and examined for light-induced chromatid damage.
In all 3 lines,
susceptibility
cells
to
both
spontaneous
showed
a signifi
chromatid
breaks
transformation
(for p
Although these data showed that the malignant cells are
9258A
0
143(20)
206 (25)
0
0
0.023
0.100
0
0
0.007
0.020
more susceptible to light-induced
chromatid damage than
are their nonneoplastic
precursors, this increased suscep
9258B
18
143(18)
0
0.120
0
0.060
tibility
206(28)
0.020
0.600
0
0.127
179(25)
0.005
0.248
0
0.192
271 (38)
0.113
0.913
0.033
9165A
a One
0
hundred
fifty
to 200
metaphase
plates
were
0.287
analyzed
long-term
for
C
shielded;E, lightexposed.
Numbers
in
parentheses,
passage
number.
3Chromatid
from
prolonged
culture
in vitro.
In order
culture.
Since horse serum accelerates
sponta
as compared with FBS
when used as a supplement
to the chemically
defined
medium, nonneoplastic
cells grown in FBS and their malig
nant derivatives grown in horse serum could be compared
(127daysonly), and 92585.
b
result
neous malignant transformation
each variable, with the exception of 50 to 100 for 92555, 9255E
@
could
to determine the relationship between increased suscepti
bihity and malignant transformation, malignant and nonneo
plastic cell lines of common origin were compared after
Table
culturesof
breaks and exchanges
in fluorescent
light-exposed
versus shielded
mouse cells before and
transformationChromatid
after spontaneous malignant
breaks
NCTCceII
liner'
Treatment
no/cellBefore
Av. no/cell
After5415
5435
5449
Cells
of
Chromatid
.
NCTC
0.030
0.130
0.025
0.335
S
0.010
0.030
E
0.085
S
E
0.013
0.260
5415
had
been
exchanges
b
Av.
After
5―
0.005E
(I
(1
Before
7 x 10@
0.005
0.020
0
0.023
1.340
<10 e
0.020
0.270
0.002
0.020
0.953
<106
0
0.025
0.005
0.130
<10'
in
culture
for
57
days
(5
passages)
0.065
and
260
0.003
days
(30
passages), NCTC 5435 for 64 days (5 passages) and 288 days (27 passages), and NCTC
5449 for 90 days (9 passages)
and 390 days (38 passages)
(3, 15). Cells were grown
in
NCTC135with 10%FBS.
b At least 200 metaphase
plates were analyzed for each variable with the exception of
114 for 5435E after transformation and 153 and 193 for 54495 before and 5449E after
transformation, respectively.
C
Level
of
significance
for
@2 test
comparing
the
frequency
distributions
of
the
chromosome abnormality in exposed cells before and after transformation.
(I
MARCH
@,shielded;
E, light
exposed.
1979
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931
R. Parshad et a!.
Table 4
Chromatid breaks and exchanges in fluorescent light-exposed versus shielded cultures of paired
nonneoplastic and malignant mouse cells
cultureChromatid
of common origin after long-term
breaks
Av no I elI
c
NCTCcell Timein vi- Nonneoplastic
line pairs tro (days) or malignant
@
pa6981
5b
186(22)@'
N
0.010
0.300
6982
238 (22)
M
0.010
0.800
5909K
319 (22@
N
0.030
0.275
5909N
297 (21)
M
0.025
0.780
7505A
7505B
0a
1013(135)
1260(169)
N
M
0.010
0
0.080
0.170
Level
of
abnormality
@
a
significance
in exposed
for
x2 test
neoplastic
shielded; E, lightexposed;
C Numbers
in
parentheses,
comparing
in Table 4 originated
pa
5
E
0
<10'
frequency
0.040
0
0.175
0.005
0.035
<10'
0
0.110
0
0.01
0
0
distributions
nonneoplastic
of
the
<10@
0.008
chromosome
counterparts.
N, nonneoplastic; M, malignant.
passage
number.
from a mouse cell line, NCTC 5405,
that was carried in culture for approximately 3 years,
remained cytologically normal, and was nontumomigenic
when repeatedly assayed in immad
iated syngeneic hosts (14).
A clone of this line was developed after 585 days in culture,
and one subline of the clone, 7505A, remained nonneoplas
tic, whereas another subline,
the
cells and their exposed
after at least 2 weeks of growth in FBS-supplemented DV
(Table 4, hines 6981 , 6982, 5909K, and 5909N). In addition,
a nonneoplastic clone and its malignant derivative, 7S05A
and 7S05B, maintained continuously in FBS-supplemented
medium were compared after at least 2 weeks of growth in
DV supplemented with FBS.
Shielded nonneoplastic and neoplastic cells of each of
the first 2 pairs were similar with respect to frequency of
chromatid breaks and exchanges (Table 4). However, the
exposed neoplastic cells of each pair were significantly
more susceptible to both chromatid breaks and exchanges
than were their nonneoplastic counterparts. The third pair
7505B, had undergone
spon
taneous malignant transformation when tested after an
additional 354 days in culture. This cloned line, which was
unusually resistant to spontaneous transformation, was
also resistant to light-induced chromosome damage. Light
exposure did not induce chromatid exchanges even in the
malignant derivative, 7505B. Further, the frequency of light
induced chromatid breaks was markedly lower than in other
mouse cell lines in spite of the longer period in culture.
However, the frequency of light-induced chromatid breaks
in the malignant derivative, 750SB, was significantly higher
than that in the nonneoplastic parental cloned line, 7505A.
DISCUSSION
A unique property of rodent and particularly mouse cells
in culture is their tendency to undergo spontaneous
neo
plastic transformation
and also to develop chromosomal
abnormalities. Although these 2 events could not be strictly
correlated, a marked increase in the frequency of abnormal
chromosomes at the time of or after neoplastic transforma
tion has been observed in a number of mouse cell lines (5,
9, 10). Three environmental factors, the type of serum
added to the chemically defined medium, fluorescent light,
932
E
Chromatid exchanges
Av
I II
. no. ce
and the oxygen concentration in the gaseous phase of the
culture, have been shown to influence both chromosome
stability and the occurrence of neoplastic transformation in
mouse cells (5, 9, 11, 16). The relationship between spon
taneous neoplastic transformation and chromosomal ab
normalities is also suggested by the stable karyotype of
human cells in culture, which thus far have not been
reported to transform spontaneously.
We have suggested that the molecular lesion underlying
the chromatid damage might be DNA or DNA-protein cross
links, which are also produced by fluorescent light (6).
However, the exact molecular mechanism needs clarifica
tion. The light-induced chromatid damage results from the
production of H@O2
within the cell (13). This photoproduct
is known to perturb
DNA (1), and some of these perturba
tions may initiate neophastic transformation. Direct pertum
bation of DNA by bromodeoxyuridine incorporation fol
lowed by near-UV light exposure has recently been reported
to result in the neoplastic transformation of Syrian hamster
embryo cells in culture (4).
Although the mechanism of spontaneous transformation
of mouse cells in culture is still unknown, the present
observations suggest that increased susceptibility to light
induced chromosome damage may be associated with or
requisite
for such transformation.
In all 3 hines followed
during continuous culture that either became tumorigenic
or showed cytological evidence of neoplastic change, there
was an associated increase in susceptibility to light-induced
chromosome damage. Increased susceptibility was also
observed in 3 cell lines after malignant transformation, as
compared with their nonneophastic precursors. The in
creased susceptibility in these lines is not simply the result
of prolonged culture, because nonneoplastic lines after
long-term culture were significantly less susceptible than
their neoplastic counterparts. In addition, the cloned line
which was most resistant to spontaneous transformation
through the years was also the most resistant to light
induced chromosome damage. Thus, it appears that the
heightened susceptibility eventually observed in these cells
is an event concomitant or associated with their sponta
neous neoplastic transformation in culture.
This increased susceptibility could result from impaired
CANCER RESEARCHVOL. 39
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Susceptibility
DNA repair capacities or from the loss or inactivation of
enzymes such as catalase or superoxide dismutase which
break down H@O2
or scavenge free radicals associated with
H@O2
production.
to Chromosome
Damage
normal adult bovine and ovine origin. Proc. 5oc. Exp. Biol. Med., 98:
574-576, 1958.
9. Parshad,R., and Sanford,K. K. Effect of horseserum,fetal calf serum,
calf serum, bovine serum, and fetuin on neoplastic conversion and
chromosomes of mouse embryo cells in vitro. J. NatI. Cancer Inst., 41:
767-779, 1968.
10. Parshad, R., and Sanford, K. K. Search for genetic influences on
neoplastic transformation
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933
Increased Susceptibility of Mouse Cells to Fluorescent
Light-induced Chromosome Damage after Long-Term Culture
and Malignant Transformation
Ram Parshad, Katherine K. Sanford, Robert E. Tarone, et al.
Cancer Res 1979;39:929-933.
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Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1979 American Association for Cancer Research.