[CANCER RESEARCH 33, 362 369, February 1973]
DNA Repair with Purines and Pyrimidines in Radiation- and
Carcinogen-damaged Normal and Xeroderma
Pigmentosum Human Cells1
J. E. Cleaver
iMboratory of RaJiobiology, University of California, San Francisco, California 94122
SUMMARY
Repair replication of DNA in cells exposed to UV, X-rays,
or chemical carcinogens involves incorporation of both purine
and pyrimidine precursors. Previous reports stating that
purines are not involved in repair must be due to insufficient
resolution in the reported experiments. Hypoxanthine was
more efficiently incorporated by repair replication than other
precursors such as adenine, deoxyadenosine, and guanine.
Hydroxyurea did not inhibit hypoxanthine incorporation by
repair replication. Cells from patients with the hereditary
high-cancer disease xeroderma pigmentosum show reduced
repair after exposure to UV, 4-nitroquinoline
1-oxide, and
1,3-bis(2-chloroethyl)-l-nitrosourea;
they show normal repair
after exposure to agents such as jV-methyl-TV'-nitro-j'v'-nitrosoguanidine. These results allow agents to be classified according
to whether or not xeroderma pigmentosum cells respond
normally to them and illustrate the values and limitations of
the use of xeroderma pigmentosum cells or measurements of
DNA repair as test systems for carcinogens.
INTRODUCTION
Radiation- and chemically induced changes in DNA have
been suggested as being involved in mechanisms of radiation
(12, 14) and chemical (2, 5, 40) carcinogenesis. In the
hereditary skin disease XP2 (31, 34), carcinogenesis from UV
may be associated with reduced levels of DNA repair (8,9, 12,
17). Among a family of nitroquinoline
derivatives with
differing carcinogenicity, higher levels of repair are induced by
the chemicals with greater carcinogenicity (38,40). In both of
these examples it seems that greater damage to DNA may be
associated with higher oncogenesis.
Current concepts of 1 repair process in mammalian cells
envisage the removal of damaged regions of DNA and synthesis
of replacement regions by incorporation of new bases (see
'Work performed under the auspices of the U. S. Atomic Energy
Commission.
'The
abbreviations used are: XP, xeroderma pigmentosum
homozygote; BrUdR, bromouracil deoxyriboside; FUdR, fluorouracil
deoxyriboside; MMNG, ;V-methyl-jV'-nitro-jV-nitrosoguanidine; 4NQO,
4-nitroquinoline 1-oxide; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea;
AdR, adenine deoxyriboside.
Received August 21. 1972; accepted November 2, 1972.
362
Refs. 12, 14, and 15 for recent reviews). This concept requires
that the replacement regions are faithful copies of the original
base sequences, but several experimental results have cast
doubt on this. For both UV- and carcinogen-damaged cells, the
incorporation of pyrimidine but not purine bases could be
detected during repair of DNA (24, 37). Since the repaired
regions, at least for UV damage, are of the order of 100 bases
(8, 14, 15, 18, 32), such observations, if true, require some
réévaluation
of the current concept of DNA repair. I have
therefore investigated repair of UV, X-ray, and carcinogen
damage in several human cell types with a variety of purine
and pyrimidine precursors. This study has taken advantage of
the known defect in repair in most forms of XP cells (4, 8, 9,
12, 17, 36) and the insensitivity of repair to inhibition by
hydroxyurea
(10) as tests of whether
the observed
incorporation of bases has the characteristics expected for
repair. The results show that both purine and pyrimidine bases
are used as precursors for repair of radiation and carcinogen
damage
to DNA. The incorporation
of thymidine,
hypoxanthine,
and adenine
can be readily detected.
Deoxyadenosine and guanine are poor precursors for both
semiconservative and repair replication. The ability of XP cells
to repair some forms of damage but not others allows some
implications to be derived for the use of these cells, and for
repair mechanisms in general, in relation to investigations of
carcinogenesis.
MATERIALS AND METHODS
Cell cultures from skin biopsies of XP patients
XP17, XP19), a parent (XPH19), normal individuals,
established culture of HeLa S3 cells were grown in
minimal essential medium with 10% fetal calf serum
(XP12,
and an
Eagle's
at 37°.
One of the patients (XP17) had the neurological symptoms of
the de Sanctis-Cacchione syndrome (31).
DNA repair was assayed in isopycnic CsCl gradients with a
method (Chart 1) based on the one first described for bacteria
(29) and since used extensively for mammalian cells (8, 12,
15). Cultures were grown for 1 hr in nonradioactive BrUdR, (3
Mg/ml) plus 1 /UM FUdR to begin the formation of
bromouracil-substituted
DNA of increased density (1.751 g/ml
in CsCl at neutral pH, in comparison to 1.700 g/ml for native
DNA). Cultures were rinsed once in 0.9% NaCl solution,
irradiated with UV (13 ergs/sq mm/sec, low-pressure mercury
germicidal lamps, predominantly 254 nm) or X-rays (300 kVp,
CANCER RESEARCH VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
DNA Repair with Purines and Pyrimidines
DNA
during
SEMICONSERVATIVE
broken
isolation
DMA REPLICATION
NORMAL
DENSITY,
(l 700gM)
DENSE
H BASE
etc
\
REPAIR
NORMAL
DENSITY
REPLICATION
(l.ZOOgjUl)
( -M-
Chart 1. Principle of experimental
semiconservative and repair replication.
UV lesions
)
scheme for labeling cells with BrUdR plus a 3H-Iabeled DNA precursor to discriminate between
700 R/min), and grown for 4 hr in medium containing FUdR
and/or BrUdR (3 /Jg/ml) plus 3H-labeled thymidine, adenine,
guanine, hypoxanthine, or deoxyadenosine (see chart captions
for concentrations
and specific activities).
In some
experiments hydroxyurea (10 mM) was also added after
radiation.
For X-rays, the labeled medium was added at the start of
radiation; cultures were then radiated at room temperature
and returned to 37° for 4-hr growth. In studies with
carcinogens, these were added at appropriate concentrations to
the medium containing BrUdR and FUdR 1 hr after the
medium was added to cultures. Incubation continued for 1 hr
before the cultures were rinsed, and the medium was replaced
with radioactively labeled medium. The carcinogens used were
MNNG, 4NQO, and BCNU. MNNG was dissolved in 0.1 M
phosphate buffer at pH 7.0, and the others were dissolved in
absolute ethyl alcohol before being added to culture media.
At the end of the labeling period, cells were harvested and
DNA was isolated and analyzed in isopycnic CsCl gradients (at
pH 7.0 for double-stranded
DNA and pH 13.0 for
single-stranded DNA) as described previously (10). The
protocol used here (Chart 1) ensures that DNA made by
semiconservative DNA replication is of increased density
through extensive replacement of thymine by bromouracil in
the daughter strands. [In the experiments with mixtures of
thymidine and BrUdR, thymidine concentration was always
about
10% of the concentration
of BrUdR. Previous
experiments
have shown that these 2 precursors are
incorporated into DNA in the proportions available in the
medium during both repair and semiconservative replication
(14)]. DNA of normal density should be labeled only if small
amounts of BrUdR and 3H-labeled precursors are incorporated
as small patches during repair replication (see Chart 1).
Radioactivity profiles from isopycnic gradients of different
cell types in a single experiment were normalized to the same
amount of DNA in the absorbance peaks to facilitate
comparison of the amounts of radioactivity incorporated.
Specific activities of normal-density DNA in alkaline gradients
were determined by pooling fractions and measuring the cpm
based on 20- to 100-min counting and absorbance at 260 nm
FEBRUARY
(an ASSO of 1.0 corresponds
specific activity is expressed
obtained from control cells.
to 50 /ng DNA per ml). The
as cpm//jg DNA above that
RESULTS
In cells untreated with radiation or chemicals, labeling with
BrUdR plus a labeled precursor of DNA produces labeled DNA
of increased density (Charts 2 to 4) as expected for
semiconservative replication (Chart 1). In alkaline isopycnic
gradients, all of the label was in dense strands of DNA that
were synthesized during the labeling period and no peaks of
normal density were observed; the incorporated BrUdR
contributes to the density increase and the labeled precursor
to the radioactivity (Charts 2 to 4).
After radiation, a new pattern of incorporation was evident
(Charts 2 and 3). The amount of radioactivity incorporated by
semiconservative replication was reduced, and radioactivity
was incorporated at normal density (Charts 2 and 3) in the
manner expected for repair replication (Chart 1). In the initial
double-stranded DNA gradients (neutral pH), radioactivity at
normal density was not easily resolved, but recentrifugation of
normal-density DNA in single-stranded form clearly resolved
this for most of the precursors (Charts 2 and 3). Repair
replication after UV radiation could also be detected with
adenine and deoxyadenosine but only marginally with guanine
(Chart 3; Table 1). The specific activity of normal-density
DNA determined from these and other gradients shows that
the incorporation of 3H-labeled BrUdR, 3H-labeled thymidine,
and 3H-labeled hypoxanthine into normal-density DNA by
repair replication is increased in normal fibroblasts and HeLa
cells but not in XP cells after UV radiation (Table 1). These
observations correspond to those reported previously, in which
normal fibroblasts and HeLa cells incorporate 3H-labeled
thymidine or 3H-labeled BrUdR into small patches (repair
replication) in their DNA after radiation, and XP cells are
defective in such repair (4, 8, 9, 11—¿
13). The similarity in
results obtained with thymidine and hypoxanthine (Chart 1;
Table 1) indicates that repair replication involves the
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
363
J. E. Cleaver
detect repair of UV or chemical damage. Since 3H-labeled
AdR proved to be a relatively poor precursor for DNA in our
hands, 3H-labeled hypoxanthine was used to test the effect of
o
-o
z
s
oc
u
Z
Z
o
u
FRACTION NUMBER
Chart 2. CsCl isopycnic gradients for cells grown in BrUdR (3
for 1 hr, radiated with 260 ergs/sq mm, and labeled for 4 hr with
BrUdR (3 Mg/ml) plus 3H-labeled hypoxanthine (20 Mg/ml, 20
Ci/mmole) or 3H-labeled thymidine (TdR) (20 /jCi/ml, 20 Ci/mmole)
before DNA was harvested. I-'UdR (1 MM)was present throughout, a,
control XP cells labeled with 3H-labeled thymidine (•)or 3H-labcled
hypoxanthine (//) (") initial gradient at neutral pH;¿>,alkaline rcband
of normal-density DNA from a; c, XP (•)
or Hela (»)cells radiated with
UV and labeled with 3H-labeIed thymidine, initial gradient at neutral
pii; <i, alkaline reband of normal-density DNA from c; c, XP (•)or
HcLa (o) cells radiated with UV and labeled with 3H-labeled
hypoxanthine, initial gradient at neutral pH: /. alkaline reband of
normal density DNA from c: ordinate, 3H cpm X IO"2.
incorporation of pyrimidines and purines and that the repair
defect in XP involves reduction in the incorporation of both
kinds of bases.
The
inhibition
of
thymidine
and
hypoxanthine
incorporation into DNA by UV is different (Chart 1) because
the amount of precursor incorporated into DNA depends on
the effects of UV on both DNA replication and the
incorporation pathways. Since the incorporation pathways and
pool sizes are different for pyrimidines and purines, the
quantitative aspects of inhibition of DNA synthesis will vary
with the precursor used and possibly also with the cell type.
Effect of Hydroxyurea on Furine Incorporation by Repair
Replication. Hydroxyurea is often used as an inhibitor to
suppress semiconservative replication preferentially, thereby
increasing the resolution of repair replication (7, 10, 11). Such
experiments have usually involved labeling with pyrimidines,
but in experiments with 3H-labeled AdR in the presence of
hydroxyurea
364
several workers (24, 37) have been unable to
hydroxyurea on repair replication. Repair replication of UV
damage was only slightly reduced by hydroxyurea in contrast
to the large inhibition of semiconservative replication (Table
2).
Repair Replication with Purines after Damage by Ionizing
Radiation
or Chemical Carcinogens.
The experiments
described above indicate that purines are incorporated during
repair replication after UV radiation and that hypoxanthine
was the better of the purine precursors tested, i.e., it gave the
highest
specific
activities
(Table
1). Consequently,
hypoxanthine was used in another series of experiments to
determine whether repair of other kinds of DNA damage also
used purines. In many of these experiments DNA was analyzed
directly on alkaline isopycnic gradients without successive
rebanding.
After radiation with a relatively high dose of X-rays (20
kR), a low level of hypoxanthine
incorporation
into
normal-density DNA could be detected (Chart 4). Many
previous experiments with 3H-labeled BrUdR have shown that
only small amounts of pyrimidine precursors are involved in
the repair of DNA strands made by ionizing radiation (27, 28),
and this result with hypoxanthine
agrees with previous
experiments. The level of incorporation was too low to pursue
further a quantitative analysis of purine incorporation.
Treatment of normal and XP cells with carcinogens also
resulted in repair replication involving the incorporation of
3H-labeled hypoxanthine (Charts 5 and 6). In most cases, the
doses of carcinogens used were high enough to suppress
semiconservative replication to a low level, and the major
peaks in the isopycnic gradients correspond to repair
replication (Charts 5 and 6). As a result of the high doses, the
overall incorporation of radioactivity is low in comparison to
the previous experiments with UV light (Chart 1), but repair
replication is clearly resolved in these gradients. Both HeLa
and XP cells perform similar amounts of repair replication
after treatment with MNNG (Chart 5), whereas after treatment
with 4NQO only normal or XP hétérozygote
cell types
performed significant amounts of repair replication (Chart 5).
Repair replication after treatment with BCNU (Chart 6)
involving purine incorporation was also observed in normal but
not XP cells.
These results may be influenced to some extent by the
effects of radiation or carcinogens on pyrimidine and purine
incorporation into cellular pools, but the reduced level of
repair replication after UV radiation of XP cells relative to
normal and HeLa cells is similar (0 to 10%) with either purine
or pyrimidine precursors (Chart 1). It is unlikely, therefore,
that effects of radiations and carcinogens on precursor
pathways would mask the repair defect in XP cells completely,
but such effects must be borne in mind in evaluating these
data.
DISCUSSION
These results indicate that repair replication after radiation
or carcinogen treatment involves the incorporation of both
CANCER RESEARCH VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
DNA Repair with Purines and Pyrimidines
ADENINE
-CONTROL
•¿
uv
ADENINE
NEUTRAL
pH
ALKALINE
pH
20
10
60-
A
40
o
-05
10
X
E
20
O
-o
CN
GUANINE
Q_
U
GUANINE
NEUTRAL
1.0
pH
z
<
ALKALINE pH
20
CD
ce:
IO
I—
Z
15
Z)
O
U
10
05
10
15
10
10
05
05
20
10
FRACTION
15
O
00
CD
20
NUMBER
Chart 3. HeLa cells grown for l tu in BrUdR (3 Mg/ml), UV-radiated with 260 ergs/sq mm, and grown for 4 hr in BrUdR (3 jig/ml) plus
3H-labeled adeninc (20 |uCi/ml, 19 Ci/mmole) or 3H-labeled guanine (20 juCi/ml, 16.6 Ci/mmole) before harvesting. I-'UdR (1 ¿iM)
was present
throughout. Top left, initial gradient of DNA from cells grown in 3II-labek'd adenine; top right, alkaline reband of normal-density DNA labeled
with 3H-labeled adenine; bottom left, initial gradient of DNA from cells grown in 3H-labeled guanine; bottom right, alkaline reband of normal
density DNA labeled by 3H-labeled guanine\ordinatc, 3H cpm X IO"2.
300-
200-
z
D
O
u
100<
5
10
FRACTION NUMBER
Chart 4. Alkaline reband of normal-density DNA from initial neutral
pH gradient of normal human fibroblasts (JEC) grown in BrUdR (3
¿ig/ml),irradiated with 20 kR of X-rays, and labeled for 4 hr with
BrUdR (3 /jg/ml) plus 3H-labeled hypoxanthine (20 /jCi/ml, 20
Ci/mmole) before being harvested. FUdR (1 ¿iM)was present
throughout. Ordinate. 3H counts per 30 min.
FEBRUARY
purines and pyrimidines into DNA, as expected on the basis of
the current model of excision repair in animal cells (see Refs.
12, 14, and 15 for reviews). The results are consistent with
other observations indicating that repaired regions can involve
replacement of many more bases than those actually damaged
(8, 14, 16, 18, 32), that repaired and unrepaired DNA show
similar DNA-DNA hybridization
kinetics (25), and that
pyrimidine labeling of isostichs is similar in repair and normal
replication (23). Previous reports (24, 37) that purine
precursors are not involved in DNA repair must therefore be
attributed either to insufficient resolution in the experimental
methods or some unique features of the cell system.
The method on which reports concerning the lack of purine
incorporation were based depends on measurement of an
increase in the total incorporation of precursor per cell
(lymphocytes) when normal replication of DNA is suppressed
by hydroxyurea. This method does not characterize the form
of
precursor
incorporation,
and only
in carefully
circumscribed conditions supported by additional data (e.g.,
from autoradiographs and isopycnic gradients) is there any
certainty that the changes in precursor incorporation really
measure repair of DNA. It is possible that this method is
adequate only when 3H-labeled-thymidine
is used as a
precursor because of the unique specificity of this nucleoside
for DNA replication. Other precursors, particularly purines,
are diverted into many biochemical pathways and, even when
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
365
J. E. Cleaver
Table 1
Specific activities of repaired DNA labeled during the 1st 4 hr
after radiation with UV (260 ergs/sq mm)
In all experiments cells were incubated in BrUdR (3 jug/ml) plus 10 MM FUdR, with a
radioactivity concentration of 20 /jCi/ml. Specific activities were 3H-labeled BrUdR. 4
Ci/mmole; 3H-labeled thymidine, 20 Ci/mmole; 3H-labeled hypoxanthine, 20 Ci/mmole;
3H-labeled adenine. 19 Ci/mmole; 3H-labeled guanine, 16.6 Ci/mmole; and 3H-labeled AdR. 18
Ci/mmole. Data are expressed to 2 significant figures.
Cell
typeNormal
activity
(cpm/Mg
DNA)550,
normal)4.0,
of
17.69.80(1
XP12HeLa
BromodeoxyuridineThymidine
380
18.0,35.0260,
XP12
XP17Normal
Thymidine
ThymidineHypoxanthine
24
0120
HeLa
XP12
XP17HeLa
Hypoxanthine
130, 120
Hypoxanthine
0
HypoxanthineAdenineGuanineDeoxyadenosine
087
220
HeLaNormal
II0
1.329
XP17PrecursorBromodeoxyuridine
DeoxyadenosineSpecific
0XP(%
Table 2
Specific activities of Hela cell DNA after radiation with UV and
labeling with ^H-labeled hypoxanthine" plus hydroxyurea
activity6Total
Dose(ergs/sq
mm)00260
(mM)0
100
260Hydroxyurea
10Specific
incorporation2800
replication1.0
162.2(cpm/Mg)Repair5.8106.2,
94.5,
111.6, 105
60, 110
" At 20 MCi/ml, 20 Ci/mmolc plus 10 mM hydroxyurea, BrUdR (1.5 jug/ml), and 10 MM
1-UdR for 4 hr.
b Reported without subtraction of levels obtained in unradiated cultures.
semiconservative
replication is blocked by hydroxyurea,
the net increase in their incorporation due to repair can be
difficult to resolve. Low resolution is also suspect in these
reports because 3H-labeled AdR (24, 37), which is one of the
However, since hydroxyurea appears to block ribonucleotide
reducÃ-ase (1, 43), it might have been expected that
hydroxyurea would reduce hypoxanthine incorporation by all
forms of DNA replication. That it does not may be explained
poorest precursors for repair, was used (Table 1). Also, it is in 2 mechanisms: (a) inhibition is incomplete, and thus
possible that some of the properties of lymphocytes related to sufficient purine precursors may be available for repair to
reactions by
radiation sensitivity and DNA repair may be different from occur; (b) the nucleoside-jV-transglycosidase
fibroblasts (7, 19, 33), which may contribute to the previous which bases and deoxyribose are exchanged may act as salvage
or emergency pathways to ensure that sufficient amounts of
results on purine incorporation (24, 37).
Repair replication
with hypoxanthine
responded
to deoxynucleotide triphosphates are available for repair to be
hydroxyurea in a manner identical to that observed with completed (26).
These experiments with purine and pyrimidine precursors
thymidine (10). Hydroxyurea suppressed semiconservative
replication to a large extent but had no effect on repair indicate that repair replication can involve a replacement of
each of the DNA bases and is not a peculiar form of
replication,
as measured by hypoxanthine
incorporation
replication involving pyrimidines alone. Experiments have also
(Table 2). This is consistent with the known preferential
shown that low levels of repair are correlated with increased
inhibition of semiconservative in contrast to repair replication
when 3H-labeled thymidine or 3H-labeled BrUdR is used (10). UV sensitivity in XP cells, indicating that repair has a
366
CANCER RESEARCH VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
DNA Repair with Purines and Pyrimidines
BCNU
•¿ »JEC
0
OXP19
-|0.8
T~
o
x
l
l
i
l
o
CN
E
l
CN
5
5
M
3
tn
t—
I ,
O
U
O
in
co
•¿'-•=,
10
FRACTION
15
FRACTION
NUMBER
Chart 5. Initial alkaline isopycnic gradients of normal (JEC), HeLa,
and XP heterozygous (XPH19) or XP homozygous cells (XP17) grown
for 1 hr in BrUdR (3 Mg/ml) and 1 hr in BrUdR (3 jug/ml) plus4NQO
(12 Mg/ml) or MNNG (70 Mg/ml) and then rinsed and labeled with
BrUdR (3 Mg/ml) plus 3H-labeled hypoxanthine (30 jiCi/ml, 20
Ci/mmole). 1'UdR (1 MM)was present throughout. Specific activities of
15
NUMBER
Chart 6. Initial alkaline isopycnic gradients for normal (JEC) or XP
cells grown for 1 hr in BrUdR (3 Mg/ml) and 1 hr in BrUdR (3 Mg/ml)
plus BCNU (15 Mg/ml) and then rinsed and labeled with BrUdR (3
Mg/ml) plus 3H-labeled hypoxanthine (20 MCi/ml, 20 Ci/mmole). FUdR
(1 MM)was present throughout. Ordinate, 3H cpm X 10"'.
which agents are classified according to whether XP cells are
sensitive to them and fail to repair resulting DNA damage, 2
normal-density repaired DNA in these experiments were 13.2 cpm/Mg broad categories are obtained from results in this and other
for JEC cells, 12.6 cpm/Mg for XPH19 cells, and 1.3 cpm/Mg for XP17 papers (Table 3). One category, which includes UV, consists of
cells. Ordinate, 3H counts per 20 min X IO"2.
agents that produce DNA damage which is not repaired by XP
cells; the other category consists of agents to which normal
functional role for cell survival (11). In conclusion, therefore,
and XP cells respond identically. This classification is identical
it is relevant to ask the more general question of whether there to one that can technically be more easily derived from the
responses of E. coli uvr~ strains (21). E. coli would be an
is any relationship between damage and repair of DNA and the
unreliable test screen only if a metabolic derivative of an agent
mechanisms of carcinogenesis. Suggestions that modifications
of DNA may be related to carcinogenesis received a stimulus was the proximate carcinogen and could be produced only by
with the discovery of reduced repair of UV damage in the human cells and not by bacteria or if an agent was detoxified
high-cancer disease XP (8, 9). This has been expanded to in man (2). The categories (Table 3) do not, however, have any
identify other damage which XP cells fail to repair (Table 3). bearing on whether or not an agent is carcinogenic to normal
One must not forget, however, that in many diseases (12, 16) individuals; both categories include carcinogens. And several of
and even in some cases of XP (7) high carcinogenesis occurs in the drugs that cause damage that can be repaired by XP cells
association with normal amounts of repair replication. If the are actually in a class of supermutagens on the basis of their
exposure of cells to an agent results in the induction of DNA very high mutagenic activity (5).
Amounts of DNA Repair as a Measure of Carcinogenicity.
repair, however, this is good evidence that the agent interacts
with DNA to cause some form of damage. Two possibilities The precise amount of repair replication depends not only on
issue from these experiments: (a) XP cells might be used as a the extent of DNA damage from an agent but also on the
test system for the detection of carcinogens; (b) the amount of manner in which the damage is repaired. This is illustrated in
DNA repair might be used as a measure of the relative the large amount of repair replication detected after moderate
carcinogenicity of various agents. Neither of these possibilities doses of UV (Chart 2; Table 1) as contrasted with that after
high doses of X-rays (Chart 4). Also the amount of
is entirely satisfactory.
XP Cells as a Test System for Carcinogens. Suggestions have hypoxanthine
incorporated
by repair after carcinogen
been made to use the high sensitivity of XP cells as a criterion treatment (Chart 5) is low in comparison to that after UV
for screening carcinogens (41), but such a screening system radiation (Chart 2). The contrast between UV and X-rays is
would have little advantage and would be more difficult and even more marked if a comparison is made between the
lengthy in use than a screen based on Escherìchiacoli mutant number of bases inserted during repair replication after doses
strains (uvr~) (2), which carry genetic defects similar to those
that produce similar levels of cell killing and mutagenesis
in XP cells (4, 8, 9, 11-15, 36). If the criterion is set up by (Table 4). More repair replication is observed after UV
FEBRUARY
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
367
J. E. Cleaver
Table 3
Classification of agents or lesions in terms of response of XP cells to them
Agents involving normal repair
in XP cells
Agents involving reduced repair
in XP cells
X-rays (9, 20)
Photolyzed bromouracil (9)
Methylmethane sulfonate (13)
MNNG (13,42)
yV-Methy Initroso urea"
UV(4, 7-9, 11-13, 36)
Methoxypsoralen adduci (3)
4NQO(38.41)
Benz(a)anthracene K-region epoxide (39)
2,3-Methyl-4-nitropyridine 1-oxide (41)
.V-Hydroxy-2-acetylaminofluorene (42)
yV-Acetoxy-2-acetylaminofluorene (35, 42)
BCNU (this paper)
" Based on grain numbers for unscheduled synthesis over cells labeled with 3H-labeled
thymidine after exposure to nitrosourea, 5 or 50 Mg/ml (J. E. Cleaver, unpublished
observations).
Table 4
Approximate numbers of lesions, mutation rates, and numbers of bases
inserted by repair replication for human jibroblasts
X-rays
UV
R (30)
D0
600 single-strand breaks (22)
LesionsMutationrateRepair
2X 10-"/£>o
(6)2
replication
(total
basesinserted)100
ergs/sq mm (11)
2.5 X IO5 dimers(16,
3X
(6)10'lO'VOo
X IO3 bases
(3 bases inserted/break)
(27.28)"30
bases
(100 bases inserted/
dimer)(8,
excised
14-16, 18,32)"17)
" The value assumed for bases inserted per lesion is an order-of-magnitude value taken from
the various estimates of cited references. It is assumed that about one-half of the dimers are
excised and all of the strand breaks are rejoined.
radiation than after X-rays because UV damage involves many
more lesions per lethal and mutagenic event and its repair
requires the insertion of many more bases per lesion (Table 4).
Studies of the relationship between DNA damage, repair,
and carcinogenesis must therefore be pursued with careful
consideration of the numbers and varieties of lesions involved
in lethal and mutagenic events and their modes of repair.
Possibly the amounts of residual unrepaired damage are more
important in carcinogenesis than the amounts of repair
replication induced by carcinogens in normal and XP cells. The
amounts of residual unrepaired damage may lead to permanent
genetic changes involving mutations or viruses that contribute
to the development of malignant cells.
3.
4.
5.
6.
ACKNOWLEDGMENTS
I am grateful for the contribution of Miss K. McGrady, who
performed preliminary experiments for this paper, and for the technical
assistance of Mr. G. H. Thomas and Mr. W. Charles.
7.
8.
REFERENCES
9.
1. Adams, R. L. P., and Lindsay, J. G. Hydroxyurea: Reversal of
Inhibition and Use as a Cell-synchronizing Agent. J. Biol. Chem.,
242: 1314-1317,1967.
2. Ames, B. M. The Detection of Chemical Mutagens with Enteric
368
10.
Bacteria. In: A. Hollaender (ed.), Chemical Mutagens: Principles
and Methods for Their Detection, Vol. 1, pp. 267-282. New York:
Plenum Publishing Corp., 1971.
Baden, H. P., Parrington, J. M., Delhanty, J. D. A., and Pathak, M.
A. DNA Synthesis in Normal and Xeroderma Pigmentosum
Fibroblasts following Treatment with 8-Methoxypsoralen and Long
Wave Ultraviolet Light. Biochim. Biophys. Acta, 262: 247-255,
1972.
Bootsma, D., Mulder, M. P., Pot, F., and Cohen, J. A. Different
Inherited Levels of DNA Repair Replication in Xeroderma
Pigmentosum Cell Strains after Exposure to Ultraviolet Irradiation.
Mutation Res., 9. 507-516, 1970.
Bresler, S. E., Kalinin, V. L., and Sukhodolova, A. T. Action of
Supermutagens on the Transforming DNA of Bacillus subtilis.
Mutation Res., 15: 101-112,1972.
Bridges, B. A., and Huckle, J. Mutagenesis of Cultured Mammalian
Cells by X-Radiation and Ultraviolet Light. Mutation Res., 10:
141-151, 1970.
Burk, P. G., Lutzner, M. A., Clarke, D. D., and Robbins, J. H.
Ultraviolet-stimulated Thymidine Incorporation in Xeroderma
Pigmentosum Lymphocytes. J. Lab. Clin. Med., 77: 759-767,
1971.
Cleaver, J. E. Defective Repair Replication of DNA in Xeroderma
Pigmentosum. Nature, 218: 652-656, 1968.
Cleaver, J. E. Xeroderma Pigmentosum: A Human Disease in Which
an Initial Stage of DNA Repair Is Defective. Proc. Nati. Acad. Sci.
U. S., 63: 428-435, 1969.
Cleaver, J. E. Repair Replication of Mammalian Cell DNA: Effects
of Compounds That Inhibit DNA Synthesis or Dark Repair.
CANCER RESEARCH VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
DNA Repair with Purities and Pyrimidines
Radiation Res., 37: 334-348, 1969.
11. Cleaver, J. E. DNA Repair and Radiation Sensitivity in Human
(Xeroderma Pigmentosum) Cells. Intern. J. Radiation Biol., 18:
557-565, 1970.
12. Cleaver, J. E. DNA Damage and Repair in Light-sensitive Human
Skin Disease. J. Invest. Dermatol., 54: 181-195, 1970.
13. Cleaver, J. E. Repair of Alkylation Damage in Ultraviolet-sensitive
(Xeroderma Pigmentosum) Human Cells. Mutation Res., 12:
453-462, 1971.
14. Cleaver, J. E. Repair of Damaged DNA in Human and Other
Eukaryotic Cells. In: D. W. Ribbons, J. F. Woessner, and J. Schultz
(eds.), Nucleic Acid-Protein Interactions-Nucleic Acid Synthesis in
Viral Infection,
pp. 87-111.
Amsterdam:
North-Holland
Publishing Company, 1971.
15. Cleaver, J. E. Repair Processes for Photochemical Damage in
Mammalian Cells. Advan. Radiation Biol., in press.
16. Cleaver, J. E., Thomas, G. H., Trosko, J. E., and Lett, J. T.
Excision Repair (Dimer Excision, Strand Breakage and Repair
Replication) in Primary Cultures of Eukaryotic (Bovine) Cells.
Exptl. Cell Res., 74: 67-80, 1972.
17. Cleaver, J. E., and Trosko, J. E. Absence of Excision of
Ultraviolet-induced
Cyclobutane
Dimers
in
Xeroderma
Pigmentosum. Photochem. Photobiol., 11: 547-550, 1970.
18. Edenberg, H., and Hanawalt, P. Size of Repair Patches in the DNA
of Ultraviolet Irradiated Cells. Biochim. Biophys. Acta. 272:
361-381, 1972.
19. Gerber, G.B., and Altman, K. I. Lymphoid Organs. In: K. I.
Altman, G. B. Gerber, and S. Okada (eds.), Radiation
Biochemistry, Vol. 2, pp. 49-79. New York: Academic Press, Inc.,
1970.
20. Kleijer, W. J., Lohman, P. H. M., Mulder, M. P., and Bootsma, D.
Repair of X-ray Damage in DNA of Cultivated Cells from Patients
Having Xeroderma Pigmentosum. Mutation Res., 9: 517—523,
1970.
21. Kondo, S., Ichikawa, H., Iwo, K., and Kalo, T. Base-change
Mutagenesis and Prophage Induction in Strains of Escherichia coli
with Different DNA Repair Capacities. Genetics, 66: 187-217,
1970.
22. Lett, J. T., and Sun, C. The Production of Strand Breaks in
Mammalian DNA by X-rays: At Different Stages in the Cell Cycle.
Radiation Res., 44: 771-787, 1970.
23. Lieberman, M. W., Rutman, J. Z., and Farber, E. Thymidine
Labeling of Pyrimidine Isostichs from Human Lymphocyte DNA
during Repair after Damage with TV-Acetoxy Acetylaminofluorene
or Nitrogen Mustard. Biochim. Biophys. Acta, 247: 497-501,
1971.
24. Lieberman, M. W., Sell, S., and Farber, E. Deoxyribonucleoside
Incorporation
and the Role of Hydroxyurea in a Model
Lymphocyte System for Studying DNA Repair in Carcinogenesis.
Cancer Res., 31: 1307-1312,1971.
25. Meltz, M. L., and Painter, R. B. Non-uniform Distribution of
Repair Replication in Mouse L-Cell DNA after Ultraviolet
Irradiation. Biophys. J., 12: 12a, 1972.
26. Murray, A. W., Elliott, D. C., and Atkinson, M. R. Nucleotide
Biosynthesis from Preformed Purines in Mammalian Cells:
Regulatory Mechanisms and Biological Significance. Progr. Nucleic
Acid Res. Mol. Biol. 10: 87-119, 1970.
27. Painter, R. B. The Importance of Repair Replication for
Mammalian Cells. In: R. F. Beers, R. M. Herriott, and R. C.
FEBRUARY
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Tilghman (eds.), Molecular and Cellular Repair Processes, Fifth
International Symposium on Molecular Biology, Baltimore, June
3_4, 1971, Suppl. 1, Johns Hopkins Medical Journal, pp.
140-146. Baltimore: The Johns Hopkins University Press, 1972.
Painter, R. B., and Young, B. R. Repair Replication in Mammalian
Cells after X-Irradiation. Mutation Res., 14: 225-235, 1972.
Pettijohn, D., and Hanawalt, P. C. Evidence for Repair-replication
of Ultraviolet Damaged DNA in Bacteria. J. Mol. Biol., 9:
395-410,1964.
Puck, T. T., Morkovin, D., Marcus, P. I., and Cieciura, S. J. Action
of X Rays on Mammalian Cells. II. Survival Curves of Cells from
Normal Human Tissues. J. Exptl. Med., 706: 485-500, 1957.
Reed.W. B., Landing, B., Sugarman, G., Cleaver, J. E., and Melnyk,
J. Xeroderma Pigmentosum, Clinical and Laboratory Investigation
of Its Basic Defect. J. Am. Med. Assoc., 207: 2073-2079, 1969.
Regan, J. D., Setlow, R. B., and Ley, R. D. Normaland Defective
Repair of Damaged DNA in Human Cells: A Sensitive Assay
Utilizing the Photolysis of Bromodeoxyuridine. Proc. Nati. Acad.
Sei. U. S., 68: 708-712, 1971.
Robbins, J. H., and Kraemer, K. H. Abnormal Rate and Duration
of Ultraviolet-induced Thymidine Incorporation into Lymphocytes
from Patients with Xeroderma Pigmentosum and Associated
Neurological Complications. Mutation Res., 15: 92-97, 1972.
Rook, A., Wilkinson, D. S., and Ebling, F. J. G. Textbook of
Dermatology, Vol. 1, p. 62. Oxford: Blackwell Publications, Inc.,
1968.
Setlow, R. B., and Regan, J. D. Defective Repair of
N-Acetoxy-2-Acetylaminofluorene-induced Lesions in the DNA of
Xeroderma Pigmentosum Cells. Biochem. Biophys. Res. Commun.,
46: 1019-1024, 1972.
Setlow, R. B., Regan, J. D., German, J., and Carrier, W. L.
Evidence That Xeroderma Pigmentosum Cells Do Not Perform the
First Step in the Repair of Ultraviolet Damage to Their DNA. Proc.
Nati. Acad. Sei., U. S., 64: 1035-1041, 1969.
Smets, L. A. Ultra-violet-light-enhanced Precursor Incorporation
and the Repair of DNA Damage. Intern. J. Radiation Biol., 16:
407-418, 1969.
Stich, H. F., and San, R. H. C. Reduced DNA Repair Synthesis in
Xeroderma Pigmentosum Cells Exposed to the Oncogenic
4-Nitroquinoline 1-Oxide and 4-Hydroxyaminoquinoline 1-Oxide.
Mutation Res., 13: 279-282, 1971.
Stich, H. F., and San, R. H. C. DNA Repair Synthesis and Cell
Survival of Repair Deficient Human Cells Exposed to the K-region
Epoxide of Benz(a)anthracene. Nature New Biol., in press.
Stich, H. F., San, R. H. C., and Kawazoe, Y. DNA Repair Synthesis
in Mammalian Cells Exposed to a Series of Oncogenic and
Non-oncogenic Derivatives of 4-Nitroquinoline-l-Oxide. Nature,
229: 416-419, 1971.
Stich, H. F., San, R. H. C., and Kawazoe, Y. Increased
Susceptibility of Xeroderma Pigmentosum Cells to Chemical
Carcinogens and Mutagens. Mutation Res., 17: 127-137, 1973.
Stich, H. F., San, R. H. C., Miller, J. A., and Miller, E. C. Various
Levels of DNA Repair Synthesis in Xeroderma Pigmentosum Cells
Exposed
to
the
Carcinogens
7V-Hydroxy
and
yV-Acetoxy-2-acetylaminofluorene. Nature, 238: 9-10, 1972.
Young, C. W., Schochetman, G., and Karnofsky, D. A.
Hydroxyurea-induced
Inhibition
of
Deoxyribonucleotide
Synthesis: Studies in Intact Cells. Cancer Res., 27: 526-534,
1967.
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
369
DNA Repair with Purines and Pyrimidines in Radiation- and
Carcinogen-damaged Normal and Xeroderma Pigmentosum
Human Cells
J. E. Cleaver
Cancer Res 1973;33:362-369.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/33/2/362
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.