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[CANCER RESEARCH 44, 5577-5582, December 1984]
Chromosomal Radiosensitivity of Human Tumor Cells during the G2
Cell
Cycle Period
Ram Parshad, Raymond Gantt, Kathenne K. Sanford,1 and Gary M.
Jones
Department of Pathology, Howard University College of Medicine,
Washington, DC 20059 /ft P.), and Laboratory of Cellular and
Molecular Biology, National CancerInstitute, Bethesda, Maryland
20205 [R. G„K. K. S., G. M. J.)
ABSTRACT
Thirteen Å“il lines derived from human tumors of diverse
tissueorigin and histopathology were compared with 12 lines of
normalskin fibroblasts with respect to chromatid damage induced
by25, 50, or 100 R of X-irradiation during the G2 period of the
cell
cycle. Only cells in metaphase were examined, and these hadbeen
irradiated 1.5 hr before fixation. When irradiated underidentical
conditions, the tumor cells showed significantly morechromatid
breaks and gaps than did the normal cells at all dosestested. The
data suggest that the increased G2 chromosomalradiosensitivity of
the tumor cells is associated with deficientDMA repair during the
G2-prophase period of the cell cycle.
INTRODUCTION
Improved banding techniques for the study of human chromosomes
have revealed an association between chromosomalaberrations and
cancer (1, 27). The chromosomal aberrationsare manifestations of a
genetic instability associated with themalignant potential of
cells. This concept is supported by theobservation that cells from
individuals genetically predisposed toa high risk of cancer exhibit
chromosome instability or hypersen-
sitivity of the genome to environmental mutagenic agents
(7).Furthermore, both mouse and human cells that have
undergonemalignant transformation in culture, when compared with
theirnormal controls, show increased chromatid damage after
exposure to visible light or X-irradiation during the G2 period of
thecell cycle (17,19, 21). Addition of the DNA repair inhibitors
ara-C2 or caffeine directly after irradiation of the cells in G2
signifi
cantly increases the incidence of chromatid damage in the
normalcells of both mouse and human origin but has little or no
influenceon the malignant cells (17,19, 21). These observations
suggestthat the normal cells have ara-C and caffeine-sensitive
DNArepair mechanisms operative during G2-prophase that are
absent
or deficient in their malignant derivatives. A process for
repair ofDNA damage late in G2 that is sensitive to inhibitors of
DNAsynthesis or repair, such as caffeine, hydroxyurea, or
aphidicolin,and that leads to chromatid aberrations has been
suggestedfrom studies with human lymphocytes (10, 14) and
Chinesehamster fibroblasts (16,24).
To determine whether enhanced G2 chromosomal radiosensitivity is
a general phenomenon associated with human neoplasia,we compared
the cytogenetic responses of cell lines derivedfrom 13 human tumors
with 12 cell lines of normal tissue origin.All tumor cell lines,
irrespective of the tissue of origin or histopathology of the
tumor, when compared with the normal cells,
1To whom requests for reprints should be addressed.2The
abbreviations used are: ara-C, 1-/i-D-arabinofuranosylcytosine;
NCI, Na
tional Cancer Institute.Received March 5,1984; accepted August
24,1984.
showed a significantly higher incidence of chromatid
aberrationsfollowing X-irradiation during G2.
Although the increased incidence of chromatid aberrations inthe
tumor cells could result from deficient DNA repair during
G2-prophase, such an increase could also result from greater
susceptibility to radiation-induced DNA damage or to a
differentialradiation-induced delay in the progression of cells
through the
G2 prophase period; this delay would influence the time
availablefor DNA repair before metaphase. Therefore, we have
evaluated,in asynchronous cell populations, the relative
susceptibility oftumor and normal cells to DNA breakage by
X-irradiation andhave compared the cells with respect to the
X-irradiation-induced
G2 block and rate of flow ol cells into metaphase.
MATERIALS AND METHODS
Cell Culture, X-lrradiation, and Cytogenetic Procedures. Unes
of
normal skin fibroblasts were obtained from the American Type
CultureCollection, Rockville, MD (CRL), The Institute for Medical
Research,Camden, NJ (GM), and from Dr. T. Kakunaga (KD) (8) and R.
Trimmer(RJH 4) of this laboratory (NCI). Tumor cell lines were
obtained from theNaval Biosciences Laboratory, Oakland, CA and had
been establishedwith support from NCI under auspices of the Office
of Naval Researchand Regents of the University of California. Three
additional lines wereprovided by Dr. S. Aaronson of this laboratory
(NCI).
Stock lines were grown in Dulbecco's modified Eagle's medium
sup
plemented with 10% fetal bovine serum (Flow Laboratories, Inc.,
Rockville, MD) in T-25 plastic flasks. Cultures were split, usually
1:2, at weekly
intervals by dispersion with trypsin. Medium was renewed 3
timesweekly, and cultures were gassed with a humidified mixture of
10% COain air. Stock cultures and medium in our laboratory were
never exposedto light of wavelength
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CHROMOSOMAL RADIOSENSITIVITY OF HUMAN TUMOR CELLS
RESULTS
X-Ray-mduced Chromatid Damage in Normal and TumorCells.
X-irradiation of mammalian cells in Gz phase just prior to
mitosis induces chromatid breaks and gaps, apparent at the
firstposttreatment metaphase. The incidence of such
aberrationsfollowing exposure of cells to low-level X-irradiation
(25,50, and
100 R) is summarized in Chart 1. In spite of the different
tissuesof origin and histopathologies, cells from all 13 tumors
showeda significantly higher incidence of radiation-induced
chromatid
breaks and gaps than did the 12 lines of normal cells
derivedfrom individuals ranging in age from 1 to 66 years. At each
dose,p < IO"6 for breaks or gaps in the tumor cell lines as a
group
and, for each tumor line individually, P < 0.002 for breaks
and
-
120
(O100
2 80
60
40
UJ 20
Gz CHROMOSOMAL RADIOSENSITIVITY OF HUMAN TUMOR CELLS
1001 ! , 1 , r-
C]BREAKS•GAPS
.10.5 1.0 0.5 1.0
HOURS AFTER X-IRRADIATION
Chart 3. Influence of time after X-irradiation (100 R) on
chromatic) damage innormal (RJH 4) (toft) and tumor (A172) (right)
cells. In the first variable, Colcemidwas added for 0.5 hr
immediately after irradiation whereas, in the second, themedium was
renewed following irradiation, and the cultures incubated for 1 hr,
andthen Colcemid was added during the last 0.5 hr of incubation. In
cultures of RJH4, 244 and 94 metaphase cells were examined at 0.5
and 1 hr, respectively, andin cultures A172,78 and 76 metaphase
cells, respectively.
an increased incidence of both chromatid breaks and gaps wasseen
in the tumor cells during the corresponding period (Chart3).
X-Ray-induced DNA Strand Breaks in Normal and Tumor
Cells. The susceptibility of normal cells (RJH 4) as
comparedwith tumor cells (A172) was evaluated by determination of
therelative molecular weights of DNA in the 2 cell
populationsimmediately after irradiation. The élution rates of the
DNA wereessentially the same for both cell lines (Chart 4). These
resultsindicate that X-irradiation caused an equivalent number of
DNA
strand breaks and that the 2 populations were similar in
theirsusceptibility to irradiation as measured by DNA
single-strand
breaks.Influence of X-lrradiation on Progression of G2 Cells
into
Metaphase. As noted earlier, radiation might differentially
delaythe progression of cells into metaphase and thus alter the
timeavailable for repair. In both normal (RJH 4) and tumor
cells(A172), X-irradiation produced, within 0.5 to 1 hr, a similar
decline
in the number of cells entering metaphase, and both cell
linesshowed an equivalent G2 block by 1.5 hr postirradiation
(Chart5). It may be noted that only a small fraction of cells
entermetaphase at 1.5 hr postirradiation. The data in Chart 1,
however, represent the chromatid damage in metaphase cells
accumulated by Colcemid during the preceding 1.0 hr, i.e., from
0.5to 1.5 hr after irradiation.
DISCUSSION
A major finding of this study is that cells derived from
13tumors of diverse tissue origin and histopathology all have
anenhanced G2 chromosomal radiosensitivity as compared with
80
60
UJCC
40
30
20
153 6
FRACTION NUMBER
12
Chart 4. DNA alkaline eiution profiles of (»cultivated normal
(RJH 4) and tumorcells (A172) with and without 500 R of
X-irradiation. O and D, unirradiated normaland tumor cell DNA,
respectively; •and •irradiated normal and tumor cell
DMA,respectively.
normal cells. A similar enhanced G;>chromosomal
radiosensitivityhas also been reported in mouse and human cells
transformedin culture (17, 19, 21) and in skin fibroblasts from
individualsgenetically predisposed to a high risk of cancer (3, 12,
18, 20,26). It is thus possible that G2 chromosomal
radiosensitivity maybe a precondition or an initiating change in
the carcinogenicprocess, though a causal relationship has not yet
been established.
The increased incidence of radiation-induced chromatid dam
age in the tumor cells as compared with the normal cells
couldresult from greater initial DNA damage by irradiation, from
adecreased radiation-induced G2 block, allowing less time for
DNA
repair (15, 28), or from an impaired capacity to repair the
DNAdamage. Initial DNA damage by irradiation was determined
inasynchronous cell populations by measuring the extent of
DNAsingle-strand breakage immediately after X-irradiation. Such
single-strand breaks provide a measure of cell susceptibility or
the
capacity of cells to cope with the free radicals generated
withinthe cell by ionizing radiation. Our results from alkaline
eiution ofDNA immediately after irradiation show that the tumor
andnormal cells are equally sensitive to DNA single-strand
breakageby X-irradiation.
With respect to a possible differential effect of irradiation
ontraverse of the cells through G2-prophase, both normal (RJH
4)
and tumor (A172) cell populations showed a similar G2
mitoticblock between 1 and 2 hr postirradiation. Furthermore,
thepercentage of irradiated cells, relative to nonirradiated,
entering
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G2 CHROMOSOMAL RADIOSENSITIVITY OF HUMAN TUMOR CELLS
coOte.
o111
5
i7
e*
100
80
60
40
20
-O- RJH-4-*- A-172
HOURS POST EXPOSURE
Charts. Influence of X-irradiation (100 R) on progression of G?
cells intometaphase. Each determination is based on 3 to 4
irradiated and 2 to 3 nonirra-diated control cultures and
represents the ratio of mean number of metaphasecells per culture
in X-irradiated to nonirradiated controls. Cells were arrested
atmetaphase by Colcemid for 0.5 hr. Bars, standard error of the
ratio of the means.
metaphase in both tumor and normal cell populations declinedat a
similar rate following irradiation (Chart 5). The cells examinedfor
chromosome damage were accumulated by Colcemid from0.5 to 1.5 hr
postirradiation and, therefore, did not encounter theblock.
It appears, therefore, that the increased incidenceof
radiation-induced chromatid damage in the tumor cells as compared
withthat in normal cells does not result from a difference in
initialsusceptibility or from a differential radiation-induced G2
block.The enhanced incidence in the tumor cells could result
fromdeficient repair of the X-ray-induced damageduring
G2-prophase.Considerable experimental evidence indicates that
chromatidaberrations result from unrepaired DNA damage; DNA
repaircapacity and extent of chromatid damage observed
followingradiation or treatment with chemical mutagens tend to be
inversely related (2, 4, 9,17, 20, 22, 26). For example, cells
fromindividuals deficient in DNA repair show more chromatid
damagefollowing treatment with DMA-damaging agents than do
cellsfrom normal individuals (3, 12, 20, 23). Furthermore, in
repair-competent normal cells, known inhibitors of DNA repair, such
asara-C or caffeine, increase the induced chromatid damage (17-19,
21, 22; Chart 2). According to the mononeme theory ofchromatid
structure, each chromatid contains a single continuousDNA double
strand. Therefore, chromatid breaks seen in the firstmetaphase
following G? irradiation represent unrepaired DNAdouble-strand
breaks. Radiation-induced chromatid gaps apparently result from
unrepaired DNA single-strand breaks (2, 13,18). The DNA breaks
could arise directly or indirectly as a resultof repair processes
(2,4,9,21,22). The results with the inhibitorof DNA repair, ara-C,
indicate that radiation-induced chromatidgaps result from DNA
damage that is dose-related and efficientlyrepaired in normal cells
in the absence of ara-C (Chart 2). ara-C
is thought to inhibit polymerase activity in nucleotide
excisionrepair. In accordance with this interpretation, the
chromatid gapsseen within 1.5 hr after irradiation result from
incomplete excisionrepair of damage produced by X-irradiation
during G2.In view ofthese findings, the tumor cells appear to be
deficient in DNArepair during G2-prophase.This possibility is
further supportedby data comparing the incidence of chromatid
damage in anormal cell line (RJH 4) and a tumor cell line (A172) at
0.5 and 1hr postirradiation (Chart 3). In the normal cells, the
incidence ofchromatid damage decreased during the period between
0.5 and1 hr following irradiation, presumably due to DNA repair.
Incontrast, both chromatid breaks and gaps in the tumor
cellsincreased during the same postirradiation period,
presumablythrough incomplete nucleotide excision repair with
initial incisionof DNA without completion of the repair process.
This defectwould result in the accumulation of DNA single-strand
breakswhich could subsequently be converted to DNA
double-strandbreaks by single-strand nuclease (2) and be manifest
as chromatid breaks.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Robert E. Tarane. Biometry Branch,
NCI, forstatistical analysis of data.
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1984;44:5577-5582. Cancer Res Ram Parshad, Raymond Gantt,
Katherine K. Sanford, et al.
Cell Cycle Period2the GChromosomal Radiosensitivity of Human
Tumor Cells during
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