[CANCER RESEARCH 36, 1397-1403, April 1976]
DNA Excision-repair Deficiency of Human Peripheral Blood
Lymphocytes Treated with Chemical Carcinogens1
Dominic Scudiero,2 Allen Norm,3 Peter Karran, and Bernard Strauss4
Department of Microbiology, The University of Chicago, Chicago, Illinois 60637
SUMMARY
@
Human peripheral blood lymphocytes stimulated with
concanavalin A for 72 hr have a 10-fold greater capacity to
repair DNA damage induced by N-acetoxy-2-acetylamino
fluorene than do unstimulated cells. The increased capacity
of concanavalin A-activated cells to repair DNA is not ob
served after 24 hr in culture, a time at which stimulated cells
have not begun to synthesize DNA. The maximum rate of
repair synthesis obtained after treatment of stimulated cells
with the “large
patch' ‘-inducingagent, N-acetoxy-2-acetyl
aminofluorene, is twice that obtained with methyl methane
sulfonate, an agent inducing ‘
small patch―repair. The dif
ference between the maximum rates obtained with N-ace
toxy-2-acetylammnofluorene and methyl methanesulfonate is
6-fold in a human lymphoblastoid line. Unstimulated lym
phocytes show almost identical rates of repair after treat
ment with either N-acetoxy-2-acetylaminofluorene
or
methyl methanesulfonate. There is close correlation be
tween the rate of N-acetoxy-2-acetylami nofluorene-i nd uced
repair synthesis and the loss of acetylaminofluorene ad
ducts from DNA. Treatment of lymphocytes with methyl
methanesulfonate leads to degradation of cellular DNA with
the production of single-stranded regions. Such degrada
tion is not observed with N-acetoxy-2-acetylaminofluroene.
We conclude that the rate of excision repair is a function of
the capacity of cells for DNA synthesis and that lymphocytes
that do not synthesize DNA have a limited repair capacity
and cannot be used to distinguish between large and small
patchrepair.
INTRODUCTION
Normal HPBL5 carry out the reactions of excision repair
after treatment with UV, y-rays, or a variety of chemical
carcinogens (2, 12, 24, 25). The levels of repair after UV
induced damage observed with unstimulated HPBL are
lower than those observed after stimulation of cells with the
mitogen phytohemagglutmnmn (4, 17). The relative repair
capabilities of stimulated and unstimulated HPBL have
I Supported by grants from Energy Research and Development Adminis
tration [E(11-1)2040] and NIH (GM 07816, Al 121 16, CA 14599-02).
2 Present
address:
3 Trainee
in
an
00452-03. Present
Bronx, N. Y.
4 To
5 The
whom
National
for
used
Institute,
training
address:
requests
abbreviations
Cancer
immunology
Department
reprints
are:
HPBL,
NIH,
program
of Surgery,
should
human
be
Bethesda,
supported
Md.
by
NIH
Montefiore
Grant
Al
Hospital,
addressed.
peripheral
blood
lymphocytes;
BND-cellulose, benzoylated naphthoylated DEAE cellulose; Con A, concana
valin A; AAAF, N-acetoxy-2-acetylaminofluorene;
MMS, methyl methane
sulfonate; dThd, thymidine; AAF, acetylaminofluorene.
Received August 25, 1975; accepted December 22, 1975.
APRIL 1976
been estimated to differ by factor of from 1.5 to 3, but the
measurements use an autoradiographic technique that
necessarily eliminates cells in S phase and therefore may
yieldminimum values.
The availability of polyclonal mitogens and of human
lymphoblastoid lines derived from Burkitt's lymphoma pa
tients permits the comparison of lymphoid cells with differ
ing metabolic capabilities. Such cell populations used in
conjunction with the BND-cellulose method for the rapid
quantitative estimation of excision repair and the rigorous
separation of repair synthesis from semiconservative repli
cative synthesis (22) have made it possible to perform a
series of experiments designed to answer the following
questions: (a) What is the effect of increased metabolic
activity on the level of repair synthesis in populations of
HPBL? (b) How well does the rate of repair synthesis meas
ured by the BND-cellulose method correlate with other pa
rameters of excision repair such as the removal of add ucts?
and (C) Two major repair types, distinguishable by the size
of the repair synthesis ‘
‘patch―
can be observed after differ
ent treatments (21). Large patch-inducing agents such as
UV result in the insertion of about 30 times more nucleo
tides per lesion than do small patch repair-inducing agents
such as y-rays. Are the differences between ‘large
patch―and ‘small-patch―-inducing
agents demonstrable in lym
phocyte cultures?
MATERIALS AND METHODS
Cell Culture. Cell line RAJI, a human lymphoblastoid line
established from a Burkitt's lymphoma patient, was ob
tamed from Dr. P. Gerber and was maintained in Roswell
Park Memorial Institute Medium 1640, Grand Island Biologi
cal Co., .Grand Island, N. V. (GIBCO), plus 20% fetal calf
serum (GIBCO) containing penicillin and streptomycin.
HPBL were isolated from normal healthy donors by a modi
fication (14) of the procedure of Boyum (1). Cells were
cultured at 37°in 5% CO2 and 95% air in Medium 199
(GIBCO) plus penicillin, streptomycin, and 20% fetal calf
serum (GIBCO), as previously described (16). The method
we used for Con A stimulation has been published (18); in
brief, about 1.2 x i0@cells/mI were incubated with 12.5 @g
Con A per ml (Calbiochem, Los Angeles, Calif.) in medium
without fetal calf serum for 1 hr, at which time serum was
added in the appropriate amount (final concentration: Con
A, 10 pg/mI, 0.9 x io6 cells/mI). Cells were incubated in
loosely stoppered tubes in a volume of 2.5 ml, for all DNA
repair studies and DNA synthesis-monitoring experiments
(16).
1397
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D. Scudiere et al.
Chemicals. AAAF, supplied by Dr. J. A. Miller, and [9-NC]/V-acetoxy-A/-acetyl-2-aminofluorene (21 mCi/mmole), pur
chased from ICN Corporation, Chemical & Radioisotopes
Division, Irvine, Calif., were dissolved in sterile dimethyl
sulfoxide before use. MMS (Eastman Organic Chemicals,
Rochester, N. Y.) was redistilled under vacuum and was
diluted in medium immediately before use. Purified formamide (99%, m.p. 2-3°,Catalog No. 1846) was obtained from
the Mallinckrodt Chemical Works, St. Louis, Mo. Hydroxyurea (Calbiochem) was dissolved in medium before use. Cul
tures were incubated with 10 /uCi [meffty/-:lH]dThd per ml
(13 Ci/mmole or 43 Ci/mmole, Schwarz/Mann, Orangeburg,
N. Y.) for the repair experiments.
BND-Cellulose Chromatography. Cell lysates were pre
pared as previously described (22). Cells were lysed with
sodium dodecyl sulfate, treated with RNase and Pronase,
and sheared by passage 5 times through a 20-gauge needle.
The BND-cellulose method of studying repair synthesis has
been previously discussed in detail (22). A slurry of BNDcellulose (Gallard-Schlesinger, Carle Place, N. Y.) was
washed, suspended in 0.3 M buffer (0.3 M NaCI, 10 4 M
EDTA, and 0.01 M Tris-HCI, pH 7.5), and approximately 0.75
g of resin in 5 ml of buffer was allowed to settle in a 5-ml
plastic syringe containing a glass fiber filter at the bottom.
After subsequent washing of the column, cell lysates in 0.3
M buffer (5 ml total volume) were adsorbed to the column.
Columns were step-eluted with 10 ml each of 0.3 M buffer, 1
M buffer (1.0 M NaCI, 10~JM EDTA, and 0.01 M Tris-HCI, pH
7.5), and 50% formamide in 1.0 M buffer. Native doublestranded DNA is eluted with 1.0 M buffer, and singlestranded DNA or double-stranded DNA containing singlestranded regions is eluted with 50% formamide-1.0 M NaCI
buffer.
Measurement of Repair. In the presence of hydroxyurea,
an inhibitor of semiconservative DNA synthesis, the pro
gression of DNA chains is slowed and isotope incorporated
by residual replicative synthesis will remain in the growingpoint region. Because the growing point contains singlestranded regions (23), DNA containing this newly incorpo
rated isotope will adhere to the column and will require
formamide for elution. Isotope incorporated by repair syn
thesis into the bulk of the DNA wil be eluted with native DNA
in the 1.0 M NaCI eluate. After corrections for pool size are
made, the specific repair activity induced by any treatment
can be calculated from the radioactivity and absorbance at
260 nm of the NaCI eluate. Our general repair protocol is:
30-min preincubation with 10 mw hydroxyurea: 60-min in
cubation with ['H]dThd, hydroxyurea, and drug; cell lysis,
and BND-cellulose Chromatography. The specific activity of
repair is determined for a standard 1-hr incubation time. We
have demonstrated that radioactivity incorporated into the
NaCI eluate following drug treatment is exclusively the re
sult of repair synthesis (22). The residual DNA synthesis in
the presence of 10 HIM hydroxyurea (measured by [:'H]dThd
incorporation into Con A-stimulated HPBL or the RAJI lymphoblastoid line was less than 2% in these experiments.
Since the levels of repair in HPBL are so much lower than
those observed in growing cells, it is desirable to use dThd
of relatively high specific activity to obtain a significant
amount of incorporation of label. To compare the results of
1398
experiments carried out with different specific activities of
dThd, it is necessary to convert all the data to a single
specific activity. Such calculations depend on the effective
pool sizes of dTTP inside the cell at the time of treatment.
We have checked the effective pool sizes in stimulated and
nonstimulated HPBL inhibited with 10 mw hydroxyurea at
various dilutions of the specific activity of the [:'H]dThd
added, as described by Scudiere et al. (22). The calculated
effective pool size varied from 1 to 1.86, depending on the
time after lymphocyte isolation and whether or not cells
were stimulated (Table 1). For example, the correction from
a specific activity of 43 Ci/mmole to 13 Ci/mmole is done by
multiplying the cpm obtained at 43 Ci/mmole by 13/43,and
then multiplying the result by the effective pool size for that
particular physiological state.
Measurement of Excision. Stimulated or unstimulated
HPBL and RAJI cells were treated with [I4C]AAAF for 15 min
at 37°.The cells were washed with medium and harvested or
incubated for up to 5 hr. After incubation, cells were
washed, harvested, and lysed with sodium dodecyl sulfate.
Lysates were treated with RNase and Pronase, and after
extensive dialysis, banded on neutral CsCI gradients and
spun to equilibrium at 30,000 rpm for 60 hr. The amount of
radioactivity bound per unit of DNA was then determined.
Biochemical Techniques. Radioactivity was determined
by precipitation of the DNA as previously described (23).
Neutral CsCI gradients were analyzed as previously re
ported (10).
RESULTS
AAAF induces a large patch DNA repair in which approxi
mately 80 to 100 nucleotides are replaced per lesion, as
compared to MMS, which induces a typical small patch
Table 1
The effective relative dThd pool size in lymphocytes incubated in
the presence of hydroxyurea
The effective pool size was measured as described in Scudieroef
al. (22) Lymphocytes were preincubated for 30 min with 10 rnw
hydroxyurea. [-'H]dThd (43 Ci/mmole) was diluted with different
concentrations of unlabeled dThd, and cells were incubated for an
additional 60 min in the presence of hydroxyurea and 10
MCi[3H]dThdper ml. The cells were then harvested and washed, and
the total acid-insoluble radioactivity was determined. The effective
pool size was calculated as follows. If O is the ratio of radioactivity
incorporated at Specific Radioactivities 1 and 2 (cpm,/cpm2) and 7",
is the concentration of dThd at Radioactivity 1 and T2is the concen
tration at Radioactivity 2, then the effective pool size of dThd will be
given by:
P =
T, - TtQ
Q-l
In our calculations, we set T, equal to 1 so that the pool size, P, is
given in relative terms.
Time in culture
(hr)24
2472
72
72Con
dThd
pool1.221.86
A-stimulatedNo
Yes
No
YesYes
(no hydroxyurea)Relative
CANCER
RESEARCH
1.42
0.90
0.90
VOL. 36
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Lymphocyte Repair
repair with 3 to 4 bases replaced per lesion (21). These
differences are reflected in the results of repair as measured
by BND-cellulose chromatography in which the maximum
specific repair activity, observed after AAAF treatment, is 6.4
times greater than that induced by MMS (22). The measure
ments are based on a standard 1-hr repair period during or
immediately after treatment and do not reflect the comple
tion of excision repair.
Repair replication in HeLa cells irradiated with UV, a large
patch repair-inducing agent, continues for over 21 hr as
demonstrated by Edenberg and Hanawalt (6), who suggest
“That
repair replication may be rate limited by an enzyme in
the repair pathway.―We therefore determined the time
course of both MMS- and AAAF-induced repair in RAJI cells.
Within 2 hr the rate of MMS-induced repair decreased to
25% of the initial rate, whereas the AAAF-induced repair
continued at 60% of the initial rate for an additional 3 hr
(Chart 1).
The difference in response to MMS and AAAF plus the
suggested rate-limiting nature of repair in growing cells (6)
suggested a comparison of stimulated and nonstimulated
HPBL. Nonstimulated HPBL treated with either MMS or with
AAAF 24 hr after isolation and culture in Medium 199 plus
20% calf serum gave specific repair activities that were not
significantly different (Chart 2) for the 2 agents. Repair was
studied over a range of doses giving a maximum of about
7.0 ±2.4 cpm/@g for AAAF and about 12.5 ±7.6 cpm/@tgfor
120
.
MMS. The results are in contrast to the observed 6-fold
differencebetween AAAF- and MMS-induced repairseen in
RAJI cells (22). Treatment of HPBL after 72 hr in culture
INCUBATION TIME (hr)
Chart 1. Time course of repair synthesis in MMS- and AAAF-treated RAJI
cells. Exponentially growing RAJI cells were preincubated with 10 mM hy
droxyurea for 30 mm at 37@;all subsequent incubations were carried out in
the presence of hydroxyurea. The remaining treatments were: MMS, cells
treated with 264 @.tg
MMS per ml (2.4 mM) for 60 mm at 37°.After washing and
resuspension, the cells were divided into equal aliquots to which [3H]dThd
(10 @.@Ci/ml,
13 Ci/mmole) was added at hourly intervals and repair was
allowed to proceed for 1 hr in each case. The value at 1 hr was obtained from
a separatecultureof cellsto which[3HJdThd
andMMSwereaddedsimulta
neously.AAAF, cellstreated with 20 MOAAAFper ml for 15 mm at 37°,
washed
and resuspended. The hourly repair values were obtained using the same
protocol as that described for MMS.
MMS
1AAAF
•OONOF?
I -72hr
U
6
100@
ConA
“ 2
“ 3
0 Pooled
-24hr±ConA;
72 hr- no ConA
80
60
140
20@
72 hinoConA
..@::::::::@@
0
@
.1@
0
10
20
i4 AAF
30
40
Concentration(pg/m!)
50
0
,
I
100
200
u
300
I
400
I
500
MMS Concentration (pg/rn!)
Chart 2. DNA repair as a function of concentration of AAAF and MMS. Lymphocyte cultures were preincubated with 10 mM hydroxyurea for 30 mm at 37@.
The cells were then treated with AAAF or MMS for 1 hr at 37°in presence of [3H]dThd and hydroxyurea as described in “
Materials and Methods.― In AAAF
treated cells: the values for cultures incubated in presence or absence of Con A for 24 hr were indistinguishable and were therefore pooled so that each 0
representsthe mean of 2 determinations from each of 3 donors; vertical bars, standard deviation of these 6 determinations; 0, cells incubated in absence of
Con A for 72 hr. data pooled from 3 donors; closed symbols, data from cells incubated in presence of Con A for 72 hr. •,
Donor 1; •,
Donor 2; A, Donor 3.
MM5-treated cells: 0, data from 3 determinations from each of 3 donors, since the values for Con A-stimulated cells at 24 hr and unstimulated cells at 24 and
72 hr were indistinguishable; closed symbols, as indicated for AAAF-treated cells.
APRIL 1976
1399
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D. Scudiero et a!.
without stimulation produced levels of repair that were simi
lar to those observed at 24 hr (Chart 2). There was a slight
elevation of the specific activity of AAAF-induced repair,
15.1 ±4.8 cpm/@.@g
for 3 preparations compared to 7.0 ±2.4
cpm/@@g
observed at 24 hr. Lymphocytes in culture do begin
to synthesize some DNA after extensive periods in culture
even without mitogen stimulation (see Chart 3, inset), and it
may be that the slight increase in repair is related to this
synthesis. Measurement of repair by nonstimulated cultures
at 48 hr and at 7 days following isolation resulted in low
levels of repair comparable to those obtained at 24 and 72
hr.
Cells treated with AAAF or with MMS 24 hr after the
addition of Con A showed a repair activity indistinguishable
from that in nonstimulated cells incubated for the same
period. In contrast, treatment with either of the compounds
72 hr after Con A was added induced levels of repair much
greater than observed in cells not exposed to mitogen
(Chart 2). The specific activity of AAAF-induced repair cor
rected for the zero-dose control increased from 6 to an
average of 69 for 3 preparations, an increase of about 11fold. MMS-induced repair increased from a maximum of ii
in nonstimulated cells to a corrected specific activity of
29.6, a 3-fold difference. Student's t test applied to the data
from the 3 Con A-stimulated preparations at 72 hr indicated
that the AAAF- and MMS-induced repair values differed at
the 95% confidence level. The maximum levels of repair
were observed at 72 hr after stimulation (Chart 3).
Replicate measurements of repair with lymphocytes from
the same donor give closer agreement than do measure
ments with HPBL preparations from different donors, sug
gesting that the variation may indicate the different proper
ties of individual lymphocyte preparations. The ratio of re
pair activities induced by MMS and AAAF are relatively
constant from donor to donor at the maximal dose, al
though the dose-response curves for stimulated cells vary
(Chart 2). We are not able to distinguish between the varia
tion caused by uncontrolled differences in the experimental
manipulation and the intrinsic variability of the lymphocytes
themselves as causes for the differences between prepara
tions.
Removal of Bound Carcinogen. The measurement of
specific excision-repair activity by our methodology re
quires: (a) that the incorporation of dThd should not be rate
limiting; and (b) that the BND-cellulose columns separate
replicating and nonreplicating portions of the DNA with
high efficiency. Since the dThd kinase activity in unstimu
lated lymphocytes is very low (20), it could be argued that a
portion of the increase in the apparent rate of repair is
artifactual and due to a general increase in dThd kinase
activity on stimulation, or to the leakage of replicative DNA
synthesis into the NaCI eluate of the BND-cellulose column
in rapidly growing cells. We have eliminated this 2nd possi
bility by a series of density transfer experiments using the
proliferating strain RAJI in which it was demonstrated that
at the level of inhibition of DNA synthesis by hydroxyurea
used in our studies, and with these carcinogens (see “
Mate
rials and Methods―),over 90% of the residual semiconserva
tive incorporation of isotope was isolated in the formamide
eluate and virtually no DNA synthesized by semiconserva
tive processes was found in the NaCI eluate of the BND
cellulose column (22). To show that the levels of repair
observed were not determined by the ability of the cells to
incorporate dThd, we decided to study the removal of
bound AAF6 as another parameter of excision repair.
Stimulated or unstimulated HPBL and RAJI cells were
caused to react with [‘4C]AAAF
for 15 mm. The cells were
washed with medium and then harvested or incubated for 2
or 4 hr. DNA was isolated as described in “
Materials and
Methods.―Unstimulated and stimulated HPBL 24 hr after
the addition of Con A removed approximately 4% of bound
carcinogen during a 4-hr incubation following AAAF treat
ment. Cultures treated with AAAF 72 hr after Con A stimula
tion removed 35% of the initially bound AAAF in a 4-hr
period. The lymphoma line RAJI removed approximately
58% of the AAAF lesions within a similar 4-hr period (Chart
4). It is therefore clear that replication-competent cells are
able to remove significantly greater amounts of adduct from
their DNA than are replication-deficient cells. Since the
Chart 3. Relation between the maximun DNA repair synthesis in human
populations that remove the greatest amount of AAAF ad
lymphocytes and the time course of Con A stimulation. DNA repair synthesis
assayswere carried out as describedin the legendto Chart2. The maxima duct are the same ones that incorporate the greatest
shown at 24 and 72 hr are the mean values of data from the 3 donors shown in
amounts of [3H]dThd by repair synthesis, it is unlikely that a
Chart 2. The value at 48 hr is the mean of determinations from 2 donors, and
generalized increase rate of DNA degradation can account
the value at 168 hr is from a single donor. Inset, Variations in DNA synthesis
for the loss of add uct.
over the same time period. DNA synthesis was measured by a series of 6-hr
pulses of [3H]dThd (0.6 @Ci/ml,3 Ci/mmole). •,lymphocytes incubated with
10 @gCon A per ml; 0, lymphocytes incubated without Con A.
1400
6 We
do
not
distinguish
between
AAF
and
its deacylated
derivative,
amino
fluorene, in these experiments.
CANCER
RESEARCH
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VOL. 36
Lymphocyte
such increase was observed following AAAF treatment. Only
a slight shift was observed in the lymphoblastoid line RAJI
treated with MMS.
We consider that the difference between MMS and AAAF
in their initiation of DNA degradation in unstimulated cells is
significant because the experiments were carried out at
doses producing equivalent amounts of inactivation of DNA
synthesis when measured with stimulated HPBL (Chart 5).
There is no completely satisfactory method for measuring
the inactivation of unstimulated lymphocytes since they
neither proliferate nor synthesize DNA, and we therefore
assume that the relative inactivation of DNA synthesis in
E
‘I
stimulatedHPBL reflects
the relative
levelsof reactionwith
,,@
q
INCUBATION TIME (hr)
Chart 4. Excision of AAF adducts from the DNA of lymphocytes and RAJI
lymphoblastoid cells. Cells were incubated with [9-'4C]AAAFfor 15 mm at 37@,
washed, and resuspended.
Aliquots were removed after 0-, 2-, and 4-hr
incubation, and the DNA was extracted and banded on neutral CsCI gradi
ents. Specific activity of the DNA (“Ccpm/@g DNA) was determined. 0,
pooled data from lymphocytes incubated with Con A for 24 hr, and without
Con A for 24 and 72 hr, and treated with 25 .tg AAAF per ml; •,lympho
cytes incubated with Con A for 72 hr and treated with 25 @.cg
AAAF per ml;
A, pooled data from experiments in which RAJI cells were treated with 20,
50, and 100 @Lg
AAAF per ml.
The results are complicated by our observation that the
reactivity of cellular DNA with AAAF is affected by stimula
tion. Cells incubated with Con A for 72 hr bound approxi
mately 1.5 times more AAF to DNA that did unstimulated
cells. Therefore, all our data are reported relative to the
amount of AAF bound by that particular cell preparation
measured immediately after reaction.
Degradation of DNA. DNA containing regions of single
strandedness is retained by BND-cellulose columns and
requires formamide or caffeine for elution. In growing
(HEp.2) cells, the single-stranded regions are found exclu
sively at the growing point in both control and MMS-treated
cells (23). During the course of our repair experiments, we
observed a decrease in the total absorbance at 260 nm in
the 1.0 M NaCI eluate after treatment of nonstimulated HPBL
with higher concentrations of MMS. This suggested that
MMS initiated a degradative process that caused an in
crease in the proportion of parental DNA containing single
stranded regions and therefore requiring formamide for
elution. This possibility was tested by determining whether
treatment with the repair-inducing agents altered the mass
distribution of parental DNA between the NaCI and formam
ide eluates.
Cells were treated and incubated in the presence of hy
droxyurea to prevent the formation of new growing points.
The eluates from BND-cellulose were dialyzed, and the
amount of DNA in each eluate was determined by absorb
ance after banding through a neutral CsCI gradient.
A considerable increase in the percentage of DNA requir
ing formamide for elution was observed in unstimulated
lymphocyte cultures after treatment with MMS (Table 2). No
APRIL 1976
Repair
the DNA of unstimulated cells. The induction of the degra
dative process by MMS and not AAAF at equivalent levels of
reaction suggests that: (a) the degradation may be partly a
result of the chemical instability of methylated nucleotides
in DNA leading to depurination and subsequent nuclease
attack (27); and (b) the AAF adducts are removed from the
DNA by a process that does not result in the accumulation
of gaps in the DNA.
DISCUSSION
The maximal rates of repair from AAAF-induced damage
for nonstimulated and stimulated HPBL (Chart 2) and for
RAJI cells (22) are in the ratio of 1:11:33 as measured by the
BND-cellulose method. Repair rates can also be estimated
from the proportion of AAAF remaining in the DNA of cells
after 4 hr of incubation. If the loss of AAF actually proceeds
by 1st-order kinetics, then the proportion of adduct remain
ing in the DNA, C/C0, will equal e@t, where t is time and k is
the reaction rate constant. At a constant time (4 hr) the rates
will be proportional to the natural logarithm of the propor
tion of adduct
remaining
in the DNA. The ratios 0.96, 0.65,
and 0.42 represent the proportion of AAF adducts remaining
after 4 hr of incubation. These correspond to rate constants
in the ratio 1:11:21,which is similar to the BND-cellulose
data.
Only a small proportion of the unstimulated HPBL popu
lation is metabolically active. Con A stimulates 15 to 20% of
the lymphocytes at 48 hr as determined by autoradiography
(19). Most
of these
cells
will
have
divided
by 72 hr (19). In
addition, there is a subpopulation of cells that will be syn
thesizing DNA for the 1st time at 72 hr (A. Norm, unpub
lished data). Therefore we estimate that 30 to 40% of the
total population will be in the cell cycle. Since essentially all
of the RAJI population is in exponential growth under our
conditions, the lymphoblastoid cells should be 2.5 to 3 times
more active in repairing AAAF adducts than are the stimu
lated HPBL if there is a direct correlation between the ability
to repair and to synthesize DNA. The repair data are in
agreement with this hypothesis. The repair rates are also in
rough agreement with the measured 9-fold increase in the
activity of the DNA maxipolymerase activity compared to the
3.0-fold increase the minipolymerase activity induced in
HPBL by 72 hr of incubation with phytohemagglutinin (3).
Since the 1st steps in AAAF excision repair include an
endonucleolytic and exonucleolytic step, we conclude from
the failure of nonstimulated HPBL to excise AAAF lesions
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0. Scudiero et a!.
Table 2
MMS-induced degradation of lymphocyte DNA
recoveredabsorbance
TreatmentAbsorbance
in 50% form
amide1.OMNaCIamideHPBLUnstimulated24
hrUntreated
MMS,66@@g/ml
7572
hrMMS,
74Stimulated
12
57
0.27
2.44
2.6618
2.03
1.83
MMS, 330 @tg/ml1.84 0.910.42
66 @g/ml
MMS, 330 @ig/ml0.61 0.220.50
AAAF, 20 @g/ml
0.6345
withCon
A24
hrUntreated
3172
hrUntreated
AAAF, 20
@g/ml1.57
1.790.71
0.8031
1.77
0.88
MMS,330 @.tg/mI2.23 0.730.52
0.60
0.40
0.7919
25
31
MMS,270 pg/mI
2.67
1.9020
40
AAAF, 20 pg/mI
MMS, 66 @g/ml
52RAJIUntreated
AAAF, 20
@@g/ml4.91
3.98
4.421.14
30
HPBLor RAJIcells weretreated for 60 mmwith MMSor AAAF.After treatment,cultures
were harvested, lysed, and adsorbed to BND-cellulosecolumns. Column eluates were
dialyzed and banded on neutral CsCl gradients. In all experiments,the recoveryof DNA
fromthe columnswasover85%.Theamountof DNAin eachfractionfromBND-cellulose
wasdeterminedaftercentrifugationby measuringabsorbanceat 260nm.
-@
100
C
0
0
0
80
I
0
MMS
U)
C
60@
‘O
.C
I—
40@
0
ck: 20@
I—
0
0
0
00!
0.!
1.0
10.
/00
DRUG CONCENTRATION (pg/mi)
due
—
level
000
induced
Chart 5. DNA synthesis in Con A-stimulated lymphocytes after treatment
with AAAF or MMS. Cultures of lymphocytes were incubated with Con A (10
@ug/ml)for 48 hr, at which time AAAF or MMS was added. After 1 hr incuba
tion at 37°the cells were harvested, washed, and resuspended
in fresh
medium. [3H]dThd (0.6 @.tCi/ml,3 Ci/mmole) was added for a further 60 mm.
Each determination was performed in triplicate. After harvesting and wash
ing, the acid-precipitable radioactivity in the cells was determined.
that there must be a limiting nuclease activity in these cells.
Some mechanism is required to couple nuclease activity
with replicative enzymes, since, in contrast to the bacterial
polymerases, mammalian DNA polymerases are devoid of
associated nuclease activity (11).
It is possible that the relationship between repair and
replicative capability could be accounted for by changes in
the physical accessibility of the DNA to repair enzymes,
1402
perhaps
to
uncoiling
during
activation.
However,
this
explanation would not account for the difference between
AAAF- and MMS-induced repair in proliferating cells as
compared to the lack of difference in nonproliferating cells,
because one would expect the relative accessibility of the
lesions induced by reagents to be the same. The hypothesis
of physical accessibility is also at variance with the observa
tions showing mRNA synthesis by 24 hr after stimulation
(see Ref. 13), a time when there are only low levels of repair.
The ratio of repair rates for AAAF- as compared to MMS
induced lesions in RAJI cells is a maximum of 6.3 (22). Given
the difference between MMS and AAAF repair modes in
replication-competent cells, there are at least 2 questions to
be asked about the behavior of the HPBL: (a) Why is the
of
MMS-induced
by AAAF
repair
equal
in nonstimulated
to
or
cells?
greater
than
that
and (b) Why is the
level of repair induced in stimulated HPBL by treatment with
MMS the same as that observed in RAJI, whereas the level
after treatment with AAAF is lower? If the levels of incision
endonuclease are coupled to the replicative capacity of
cells, as indicated by our data, then it is reasonable that
MMS-induced damage might be repaired as rapidly as
AAAF-induced damage in nonstimulated HPBL, since the
damage induced by MMS need not require an initial incision
enzyme step because the methylated bases produced depu
rinate spontaneously (27). An increase in polymerase in Con
A-stimulated HPBL will permit repair of a greater number of
MMS-induced lesions, giving values of repair equivalent to
those found with RAJI. Since the level of incision endonu
clease is lower in stimulated HPBL than in RAJI (Chart 4)
and since this enzyme probably limits the rate of the overall
CANCERRESEARCHVOL.36
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Lymphocyte
repair process (6), AAAF-induced repair will be lower in the
HPBL than in the lymphoma line.
Finally, it might be argued that the equivalence of MMS
and AAAF-induced repair in unstimulated lymphocytes is
spurious since all lymphocytes might repair MMS-induced
damage, but only a selected few cells in the population
might be able to carry out large patch repair. However, it has
been shown by autoradiography that after treatment with
UV, a large patch-inducing agent, virtually all HPBL can
7.
8.
9.
10.
carry out at a low level of repair (8).
Our data indicate that the peripheral lymphocytes are
deficient for large patch repair because of limitations in the
levels of critical enzymes. In this respect they are similar to
other nondividing tissues such as brain, in which the exci
sion of DNA adducts either does not occur or is greatly
delayed (9, 15). This conclusion has both theoretical and
practical consequences. Lymphocytes are extremely radi
osensitive and it is reasonable to ascribe this sensitivity to
their repair deficiency and to the DNA degradation that
accompanies it. The inability of normal lymphocytes to
complement xeroderma fibroblasts (5) is also understanda
ble if lymphocytes are deficient in repair enzymes.
HPBL are used as sensitive indicators of induced chromo
somal structural change (7, 26). The ready availability of
these cells makes them a potentially excellent source of
human material with which to study the repair of DNA after
insult with putative carcinogens. However, our data suggest
that lymphocytes are very poor cells with which to investi
gate repair because of the limiting concentration of repair
enzymes. Furthermore, the extreme dependence of the level
of repair activity on the proportion of cells in the growth
cycle means that a great deal of variability in repair activities
must be expected from different cell preparations with dif
ferent numbers of proliferating cells.
11.
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1403
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
DNA Excision-repair Deficiency of Human Peripheral Blood
Lymphocytes Treated with Chemical Carcinogens
Dominic Scudiero, Allen Norin, Peter Karran, et al.
Cancer Res 1976;36:1397-1403.
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