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Regulation of repair of naturally occurring DNA strand breaks in lymphocytes
Vol.
122,
No.
July
18, 1984
1, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
REGULATION
OF REPAIR OF NATURALLY OCCURRING BREAKS IN LYMPHOCYTES
366-372
DNA STRAND
W .L. Greer and J .G. Kaplan Department of Biochemistry University of Alberta Edmonton, Alberta, T6G 2H7, Canada Received
June 8, 1984
Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD’, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 fim nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD’], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD-] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.
Recent
reports
breaks, regulated sion
(1.2.3)
by ADP ribose polymerase.
as cells differentiate;
dividing
chick
myoblasts
become multinucleated to contain nucleoid
have proposed
DNA
indeed were
this
found
muscle fibers
breaks which
sedimentation
method
that
change in the number
may be a mechanism
was anticipated to acquire
DNA
Human
peripheral
(1).
were repaired used in this
within work
for altering
breaks blood
as they
did not identify
the number
ients
following
the increased
DNA
caused by a drop
mitogen
in number
was attributed
of breaks.
Neutral
to
gradients
Rapidly
(2).
The
of breaks
in neutral
permitted
to
were reported
stimulation
sedimentation
with
(4).
differentiated
lymphocytes
8h of mitogenic
strand
gene expres-
several years before
present in resting cells; the slower observed rate of nucleoid stimulation
of DNA
supercoiling
gradof
the distinction
between strand breaks and alkali labile bonds. Using a more accurate technique shown
that about
for the estimation
3000 strand breaks per cell, in resting
Abbreviations : NAD , nicotinamide adenine dinucleotide ; LPS ) lipopolysaccharide ; TdR , thymidine
of DNA
strand breaks, we have
mouse splenocytcs, Con A,
concanavalin
were repaired A ;
Vol.
122,
within
No.
1, 1984
2h of onset of culture
resting and proliferating strand
activation. ADP
and
breaks,
AND
BIOPHYSICAL
with
(5).
Additional
mitogen
lymphocytes the steady
implicated
strand breaks during
state
number
of
these
excision repair
induced proliferation
inhibitors
of
ADP
production breaks
sequent proliferation
(2,5).
phocytes contained
This,
may be rate limiting resting lymphocytes.
for
together
Our data support
was involved
polymerase
(8)
increased
following
a substrate for ADP
ribosylation
after
of DNA mitogenic
and mouse lymphocytes
that
in the repair
prevented
of
and therefore
for
the
mitogen-
as well as their sub-
the recent demonstration
which
that
mitogen
resting lymtreatment
(9),
ribose polymerase
(10).
repair
of
strand
breaks in
this hypothesis.
Materials Cell Preparation
with
levels of NAD’. ADP
decreased
and in mouse lymphocytes,
low levels of [NAD’]
suggested to us that cellular
(7),
and repair
(2.5).
ribose
of strand breaks in human
COMMUNICATIONS
data (6) suggested that in both
there was a continuous
in efficient
mitogen
benzamide
induced repair
and Methods
and Culture
Balb/c male mice, 8-12 spleens disrupted through a wire 0.83% ammonium chloride. Cells serum, and 2 mM glutamine, at Detection
RESEARCH
Evidence was presented in the case of both human
ribosylation.
The
BIOCHEMICAL
of DNA
weeks old, were killed by cervical dislocation and their mesh screen. Red blood cells were removed by lysis with were cultured in RPM1 1640 medium with 10% fetal calf a density of 2 x 10” cells/ml.
Strand Breaks
DNA strand breaks were detected using the fluorometric analysis of DNA unwinding technique developed by Birnboim and Jevcak (11). After denaturation at pH 12.8 for 1 h. the cell lysate was adjusted to pH 11, and ethidium bromide was added. Under these conditions, ethidium bromide binds to, and fluoresces selectively in, double stranded DNA. From the percentage of double stranded DNA remaining after alkaline treatment, one can estimate the number of DNA strand breaks by using a calibration curve obtained from cells treated with various doses of gamma radiation [500 rads of y radiation produce 6000 single strand breaks per diploid genome (12)]. Assavs for ADP insoluble (13).
Ribosylation.
and NAD.
Levels
ADP ribosylation was measured as incorporation of [‘HI NAD’ into the acidfraction of permeabilized cells according to the method of Berger and Johnson
NAD’ levels were measured according to the method of Nisselbaum This method distinguishes oxidized and reduced forms of NAD.
and Green
(14).
Results In agreement of mouse
splenocytes
with
our previous
contained
DNA
report
(5),
strand
breaks which
367
Table
1 shows that a mixed population were
repaired
within
2h after
Vol.
122,
No.
1, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
RESEARCH
1
Repair of strand breaks in lymphocytes from lhymus, and “nude” spleen aper 2h treafment
Cell Source
Normal
Spleen
Thymus
Nude
%Double Stranded DNA’
Treatment
Spleen
COMMUNICATIONS
normal spleen, with mitogen
r3H] Tdr incorp./106 cells at 48h cpm
% Blasts at 48h2
Resting Con A 2 &/ml LPS 20 pg/ml
46 + 3 71 * 5 66 +I 4
5 82 46
2.400 42.300 4.800
900 6,000 1,200
Resting Con A 3 &ml + IL-23 8 units/ml
62 f
4
3
1,200
150
65 + 4
65
28,400
900
Resting Con A 2 &/ml LPS 20 ug/ml
48 Tk 4 55 f 2 56 f 3
14 17 60
1,700 2.400 11.400
600 400 2,000
‘This refers to the % double stranded DNA remaining after lh denaturation in alkali; values are inversely proportional to number of strand breaks. Using a calibration curve obtained from cells treated with various doses of y radiation, the difference in % double stranded DNA observed in spleen compared to thymus was found to represent a difference of 3000 to 4000 strand breaks per diploid genome. 2Blasts 31L2
=
mitogenic (Con
were
cell with
stimulation.
This repair
A and LPS respectively).
occurred
DNA
breaks
after treatment
of ‘H-TdR
effect of mitogen contained
diameter
larger
than
10 um.
fewer
treatment
of breaks was induced The predominantly
that
were
repaired
by either
B cells from after
T or B cell mitogens spleen of athymic
stimulation
with
LPS.
Repair
nude also
of these B cells with the T cell mitogen Con A, yet no incorpor-
occurred
Con A varied from
After
as any
Interleukin-2.
mice contained
ation
defined
at 48h. The
extent of repair
30% to 100% of that observed with on splenic and thymic
lymphocytes.
breaks per genome than those from with Con A and interleukin
in B cells after LPS.
Table
Unstimulated
incubation
with
1 also compares the cells from
the spleen of normal
2, thymocytes did not undergo
the thymus
or nude mice. further
decrease
in strand breaks, but they did proliferate. In
order
to
test
the
erase, is limiting
for ADP
the intracellular
concentration
nicotinamide with
to the external
hypothesis
ribosylation
330 to 420 PM nicotinamide
the substrate
(and thus for repair)
of NAD’ growth
NAD’,
that
resulted
Figure
in a 2-fold 368
ribose polym-
in resting splenic lymphocytes,
was varied experimentally
medium.
for ADP
1 (inset)
by adding shows
that
its precursor 2h incubation
increase in the overall intracellular
BIOCHEMICAL
Vol. 122, No. 1, 1984
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. Strand breaks were assayed after 2h incubation with various concentrations of nicotinamide in the external growth medium. Intracellular levels of NADC were measured in the same samples and are shown in the inset. There was no change in cell viability in the presence of nicotinamide.
concentration repaired
of
NAD’.
Figure
in the absence of Con
noted in the stimulated trol levels. Repair to
its
well
known
methoxybenzamide,
effect
by about
these conditions
the incorporation
strand
breaks
of thymidine
higher
not
concentrations inhibitor
occur
if
the
of ADP
ribose
ribose
DNA
10% of con-
of nicotinamide, ADP
into
were
probably
polymerase
polymerase
due (15).
inhibitor,
was also present.
2-fold
lymphocytes
(9).
stimulation,
which
that NAD’
(Figure
2).
There
was
indicates
may be limiting
Figure
(compare
was found
2)
measured
with
no
also noted
increase the rise
for repair
in in
intracellular
in permeabilized
closely
cells. Both
by Con A. 369
levels of NAD’
in the case of
nicotinamide
3 shows that the 2-fold to coincide
in resting cells was supported
Con A the intracellular
a phenomenon
that
increased transport.
repair induced
with
did
that after incubation
Fig.
under
cells at 48h was either absent or reached only
as a competitive
repair
The hypothesis by the finding
control
that
A. However,
was not induced
Nicotinamide-induced
capacity
1 shows
uptake NAD’
increase in intracellular with
peripheral
noted was
the increase in
increased
not
after the
blood Con
A
result
of
levels of NAD’ ADP
of these events precede the major
ribosylation burst of
Vol.
m w
122,
No.
I
0
1, 1984
I5
TIME
AFTER
BIOCHEMICAL
45
30 ADDITION
OF
A
(ml”
BIOPHYSICAL
m
60
Con
AND
)
TIME
w
RESEARCH
AFTER
COMMUNICATIONS
ADDITION
OF
Con
A
min1
(
Figure 2. Con A was added to cells at staggered times from 0 to 60 min. and the intracellular concentration of NAD+ was assayed in all samples at 60 min. Figure 3. Con A was added to cells at staggered times from 0 to 160 min. and ADP ribosylation was assayed in all samples at 160 min. as incorporation of PH] NAD into the acid soluble fraction of permeabilized cells.
Discussion It has been hypothesized an important
that an increase in the number
part of cell differentiation
addition
of the increase
with
the decrease in number
NAD’
and not yet fully differentiated
in cell [NAD’]
with
of DNA
the increase
strand
breaks.
levels are raised by exogenously supplied
conclude
that [NAD’]
fact
that
methoxybenzamide accords with medium
protected
that NAD’ reported
is a rate limiting
nicotinamide-induced supports
the finding human
the
for
repair
did
conclusion
lymphocytes
that (17)
against
DNA
Since this
not
repair
repair 370
since the
(16).
ribosylation
activity
in and
decrease also occurs if
in the absence of Con A, we
mitogen-induced occur involves
that nicotinamide
y radiation
for excision repair
stimulated
breaks is
there is a good correlation
in ADP
nicotinamide
factor
of KoI and Ben-Hur
may be rate limiting that nicotinamide
with the above hypothesis,
of Con A to mouse splenocytes,
time
The
consistent
of thymocytes appear to be immature, Following
strand
(3). Our data show that resting splenocytes contain
more breaks than thymocytes, an observation majority
of DNA
damage;
repair in ADP
in lymphocytes.
the
presence
ribosylation.
of This
in the external growth these authors
suggested
in these cells. Berger and Sikorski synthesis in human
lymphocytes
(18) treated
Vol. 122, No. with
UV
1, 1984
BIOCHEMICAL
radiation.
Other
activity of the ADP
did
Con A induced
athymic
nude
and/or
proceed repair
COMMUNICATIONS
in resting
lymphocytes
ligase and p polymerase,
into
further
induced
are the
and iocation
to the increase in cell [NAD-]
that follows
activation.
of the
and repair
with
stimulation
that is necessary but not sufficient
the T cell
from
the spleen of
(Table
1). We have
of DNA
strand breaks
a reduction
We conclude
in resting lymphoFurther,
blasts or proliferate
production
mitogen.
repair
B cell population
mouse splenic lymphocytes, with
DNA
stages of
in the predominantly
(6) that there is a continual
and stimulated
breaks
RESEARCH
repair
levels of NAD’
level of breaks after stimulation
strand
limiting
mice, but the cells did not form
elsewhere
in resting
cellular
not
mitogen
shown
BIOPHYSICAL
structure.
increased
cytes, the latter
factors
ribose polymerase
breaks in the DNA Although
possible
AND
in the steady state
that this is at least partly
and that this causes repair
for
lymphocyte
activation
due
of DNA
and prolifer-
ation.
Acknowledgements This
research
Engineering Greer
Research
has
been
Council
is a post-doctoral
supported
and The
fellow
by
Medical
funded
grants
from
Research
by the Alberta
The
Natural
Science
and
Council
of Canada.
Dr.
Wenda
Heritage
Foundation
for
Medical
Research.
References 1. Farzaneh, 2. Johnstone, 3. Williams.
F., Zalin, A.P., G.T..
R., Brill,
D., and Shall, S. (1982).
Nature 300, 362-366.
and Williams,
G.T.
(1982).
Nature 300. 3G8-370.
and Johnstone.
A.P.
(1983).
Bioscience Rep. 3, 815-830.
4. Scher, W., and Friend,
C. (1978)
Cancer Res. 38, 841-849.
5. Greer,
W.L.,
and Kaplan,
J.G.
(1983).
Biochem. Biuphys.
6. Greer.
W.L.,
and Kaplan,
J.G.
(1984).
Cell and Tissue Kinet. (in press)
7. Shall, S., Goodwin, P ., Halldorsson, H.. Khan, (1977). Biochem. Sot. Symp. 42, 103-116. 8. Durkacz,
B.W.,
9. Burger, N.A., 137. 79-88.
Omidiji,
O., Gray.
Berger. S.J ., Sikorski.
D.A.,
Res. Comm.
G., Skidmore,
C., and Tsopanakis,
and Shall, S. (1980).
G.W ., & Catino,
371
D.M.
115, 834-840.
Nature
(1982).
C.
283, 593-596. Exp. Cell. Res.
Vol.
122,
No.
1, 1984
BIOCHEMICAL
AND
10. Lehninger, A.L. (1976). The molecular Publishers Inc., New York. 11. Birnboim,
H.C.,
BIOPHYSICAL
RESEARCH
basis of cell structure
and Jevcak, J.J. (1981).
Cancer
Resenrch
COMMUNICATIONS
and function.
Worth
1889-1892.
41,
12. Ormerod, M.R. (1976). In Biology of Radiation Carcinogenesis (ed. J .M. Yuhas, R.W. Tennant & J.D. Rogers) Raven Press, New York pp. 67-90. 13. Berger, N.A.,
and Johnson,
E.S. (1976).
14. Nisselbaum,
J.S., and Green, S. (1969).
15. Niedergang.
C., Okazaki.
Biochim. Anal.
H.. and Mandel,
P
Biophys. Biochem.
(1979).
Acta 27,
Eur.
125,
l-17.
212-217. J.
Biochem.
102,
43-57.
16. Douglas, S.D. (1980). In Basic and Clinical Immunology (Ed. H.H. Fundenberg, D.P. Stites, J.L. Caldwell and J.V. Wells), Lange Medical Publications, Los Altos, California, pp. 96-114. 17. Kol, 18.
R., and Ben-Hur,
Berger.
N.A.,
E. (1983).
and Sikorski.
G.W.
Radiat.
Environ.
(1980).
Biochem.
372
Biophpys. Biophys.
22,
133-140.
Res.
Comm.
95,
67-72.
122,
No.
July
18, 1984
1, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
REGULATION
OF REPAIR OF NATURALLY OCCURRING BREAKS IN LYMPHOCYTES
366-372
DNA STRAND
W .L. Greer and J .G. Kaplan Department of Biochemistry University of Alberta Edmonton, Alberta, T6G 2H7, Canada Received
June 8, 1984
Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD’, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 fim nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD’], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD-] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.
Recent
reports
breaks, regulated sion
(1.2.3)
by ADP ribose polymerase.
as cells differentiate;
dividing
chick
myoblasts
become multinucleated to contain nucleoid
have proposed
DNA
indeed were
this
found
muscle fibers
breaks which
sedimentation
method
that
change in the number
may be a mechanism
was anticipated to acquire
DNA
Human
peripheral
(1).
were repaired used in this
within work
for altering
breaks blood
as they
did not identify
the number
ients
following
the increased
DNA
caused by a drop
mitogen
in number
was attributed
of breaks.
Neutral
to
gradients
Rapidly
(2).
The
of breaks
in neutral
permitted
to
were reported
stimulation
sedimentation
with
(4).
differentiated
lymphocytes
8h of mitogenic
strand
gene expres-
several years before
present in resting cells; the slower observed rate of nucleoid stimulation
of DNA
supercoiling
gradof
the distinction
between strand breaks and alkali labile bonds. Using a more accurate technique shown
that about
for the estimation
3000 strand breaks per cell, in resting
Abbreviations : NAD , nicotinamide adenine dinucleotide ; LPS ) lipopolysaccharide ; TdR , thymidine
of DNA
strand breaks, we have
mouse splenocytcs, Con A,
concanavalin
were repaired A ;
Vol.
122,
within
No.
1, 1984
2h of onset of culture
resting and proliferating strand
activation. ADP
and
breaks,
AND
BIOPHYSICAL
with
(5).
Additional
mitogen
lymphocytes the steady
implicated
strand breaks during
state
number
of
these
excision repair
induced proliferation
inhibitors
of
ADP
production breaks
sequent proliferation
(2,5).
phocytes contained
This,
may be rate limiting resting lymphocytes.
for
together
Our data support
was involved
polymerase
(8)
increased
following
a substrate for ADP
ribosylation
after
of DNA mitogenic
and mouse lymphocytes
that
in the repair
prevented
of
and therefore
for
the
mitogen-
as well as their sub-
the recent demonstration
which
that
mitogen
resting lymtreatment
(9),
ribose polymerase
(10).
repair
of
strand
breaks in
this hypothesis.
Materials Cell Preparation
with
levels of NAD’. ADP
decreased
and in mouse lymphocytes,
low levels of [NAD’]
suggested to us that cellular
(7),
and repair
(2.5).
ribose
of strand breaks in human
COMMUNICATIONS
data (6) suggested that in both
there was a continuous
in efficient
mitogen
benzamide
induced repair
and Methods
and Culture
Balb/c male mice, 8-12 spleens disrupted through a wire 0.83% ammonium chloride. Cells serum, and 2 mM glutamine, at Detection
RESEARCH
Evidence was presented in the case of both human
ribosylation.
The
BIOCHEMICAL
of DNA
weeks old, were killed by cervical dislocation and their mesh screen. Red blood cells were removed by lysis with were cultured in RPM1 1640 medium with 10% fetal calf a density of 2 x 10” cells/ml.
Strand Breaks
DNA strand breaks were detected using the fluorometric analysis of DNA unwinding technique developed by Birnboim and Jevcak (11). After denaturation at pH 12.8 for 1 h. the cell lysate was adjusted to pH 11, and ethidium bromide was added. Under these conditions, ethidium bromide binds to, and fluoresces selectively in, double stranded DNA. From the percentage of double stranded DNA remaining after alkaline treatment, one can estimate the number of DNA strand breaks by using a calibration curve obtained from cells treated with various doses of gamma radiation [500 rads of y radiation produce 6000 single strand breaks per diploid genome (12)]. Assavs for ADP insoluble (13).
Ribosylation.
and NAD.
Levels
ADP ribosylation was measured as incorporation of [‘HI NAD’ into the acidfraction of permeabilized cells according to the method of Berger and Johnson
NAD’ levels were measured according to the method of Nisselbaum This method distinguishes oxidized and reduced forms of NAD.
and Green
(14).
Results In agreement of mouse
splenocytes
with
our previous
contained
DNA
report
(5),
strand
breaks which
367
Table
1 shows that a mixed population were
repaired
within
2h after
Vol.
122,
No.
1, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
TABLE
RESEARCH
1
Repair of strand breaks in lymphocytes from lhymus, and “nude” spleen aper 2h treafment
Cell Source
Normal
Spleen
Thymus
Nude
%Double Stranded DNA’
Treatment
Spleen
COMMUNICATIONS
normal spleen, with mitogen
r3H] Tdr incorp./106 cells at 48h cpm
% Blasts at 48h2
Resting Con A 2 &/ml LPS 20 pg/ml
46 + 3 71 * 5 66 +I 4
5 82 46
2.400 42.300 4.800
900 6,000 1,200
Resting Con A 3 &ml + IL-23 8 units/ml
62 f
4
3
1,200
150
65 + 4
65
28,400
900
Resting Con A 2 &/ml LPS 20 ug/ml
48 Tk 4 55 f 2 56 f 3
14 17 60
1,700 2.400 11.400
600 400 2,000
‘This refers to the % double stranded DNA remaining after lh denaturation in alkali; values are inversely proportional to number of strand breaks. Using a calibration curve obtained from cells treated with various doses of y radiation, the difference in % double stranded DNA observed in spleen compared to thymus was found to represent a difference of 3000 to 4000 strand breaks per diploid genome. 2Blasts 31L2
=
mitogenic (Con
were
cell with
stimulation.
This repair
A and LPS respectively).
occurred
DNA
breaks
after treatment
of ‘H-TdR
effect of mitogen contained
diameter
larger
than
10 um.
fewer
treatment
of breaks was induced The predominantly
that
were
repaired
by either
B cells from after
T or B cell mitogens spleen of athymic
stimulation
with
LPS.
Repair
nude also
of these B cells with the T cell mitogen Con A, yet no incorpor-
occurred
Con A varied from
After
as any
Interleukin-2.
mice contained
ation
defined
at 48h. The
extent of repair
30% to 100% of that observed with on splenic and thymic
lymphocytes.
breaks per genome than those from with Con A and interleukin
in B cells after LPS.
Table
Unstimulated
incubation
with
1 also compares the cells from
the spleen of normal
2, thymocytes did not undergo
the thymus
or nude mice. further
decrease
in strand breaks, but they did proliferate. In
order
to
test
the
erase, is limiting
for ADP
the intracellular
concentration
nicotinamide with
to the external
hypothesis
ribosylation
330 to 420 PM nicotinamide
the substrate
(and thus for repair)
of NAD’ growth
NAD’,
that
resulted
Figure
in a 2-fold 368
ribose polym-
in resting splenic lymphocytes,
was varied experimentally
medium.
for ADP
1 (inset)
by adding shows
that
its precursor 2h incubation
increase in the overall intracellular
BIOCHEMICAL
Vol. 122, No. 1, 1984
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. Strand breaks were assayed after 2h incubation with various concentrations of nicotinamide in the external growth medium. Intracellular levels of NADC were measured in the same samples and are shown in the inset. There was no change in cell viability in the presence of nicotinamide.
concentration repaired
of
NAD’.
Figure
in the absence of Con
noted in the stimulated trol levels. Repair to
its
well
known
methoxybenzamide,
effect
by about
these conditions
the incorporation
strand
breaks
of thymidine
higher
not
concentrations inhibitor
occur
if
the
of ADP
ribose
ribose
DNA
10% of con-
of nicotinamide, ADP
into
were
probably
polymerase
polymerase
due (15).
inhibitor,
was also present.
2-fold
lymphocytes
(9).
stimulation,
which
that NAD’
(Figure
2).
There
was
indicates
may be limiting
Figure
(compare
was found
2)
measured
with
no
also noted
increase the rise
for repair
in in
intracellular
in permeabilized
closely
cells. Both
by Con A. 369
levels of NAD’
in the case of
nicotinamide
3 shows that the 2-fold to coincide
in resting cells was supported
Con A the intracellular
a phenomenon
that
increased transport.
repair induced
with
did
that after incubation
Fig.
under
cells at 48h was either absent or reached only
as a competitive
repair
The hypothesis by the finding
control
that
A. However,
was not induced
Nicotinamide-induced
capacity
1 shows
uptake NAD’
increase in intracellular with
peripheral
noted was
the increase in
increased
not
after the
blood Con
A
result
of
levels of NAD’ ADP
of these events precede the major
ribosylation burst of
Vol.
m w
122,
No.
I
0
1, 1984
I5
TIME
AFTER
BIOCHEMICAL
45
30 ADDITION
OF
A
(ml”
BIOPHYSICAL
m
60
Con
AND
)
TIME
w
RESEARCH
AFTER
COMMUNICATIONS
ADDITION
OF
Con
A
min1
(
Figure 2. Con A was added to cells at staggered times from 0 to 60 min. and the intracellular concentration of NAD+ was assayed in all samples at 60 min. Figure 3. Con A was added to cells at staggered times from 0 to 160 min. and ADP ribosylation was assayed in all samples at 160 min. as incorporation of PH] NAD into the acid soluble fraction of permeabilized cells.
Discussion It has been hypothesized an important
that an increase in the number
part of cell differentiation
addition
of the increase
with
the decrease in number
NAD’
and not yet fully differentiated
in cell [NAD’]
with
of DNA
the increase
strand
breaks.
levels are raised by exogenously supplied
conclude
that [NAD’]
fact
that
methoxybenzamide accords with medium
protected
that NAD’ reported
is a rate limiting
nicotinamide-induced supports
the finding human
the
for
repair
did
conclusion
lymphocytes
that (17)
against
DNA
Since this
not
repair
repair 370
since the
(16).
ribosylation
activity
in and
decrease also occurs if
in the absence of Con A, we
mitogen-induced occur involves
that nicotinamide
y radiation
for excision repair
stimulated
breaks is
there is a good correlation
in ADP
nicotinamide
factor
of KoI and Ben-Hur
may be rate limiting that nicotinamide
with the above hypothesis,
of Con A to mouse splenocytes,
time
The
consistent
of thymocytes appear to be immature, Following
strand
(3). Our data show that resting splenocytes contain
more breaks than thymocytes, an observation majority
of DNA
damage;
repair in ADP
in lymphocytes.
the
presence
ribosylation.
of This
in the external growth these authors
suggested
in these cells. Berger and Sikorski synthesis in human
lymphocytes
(18) treated
Vol. 122, No. with
UV
1, 1984
BIOCHEMICAL
radiation.
Other
activity of the ADP
did
Con A induced
athymic
nude
and/or
proceed repair
COMMUNICATIONS
in resting
lymphocytes
ligase and p polymerase,
into
further
induced
are the
and iocation
to the increase in cell [NAD-]
that follows
activation.
of the
and repair
with
stimulation
that is necessary but not sufficient
the T cell
from
the spleen of
(Table
1). We have
of DNA
strand breaks
a reduction
We conclude
in resting lymphoFurther,
blasts or proliferate
production
mitogen.
repair
B cell population
mouse splenic lymphocytes, with
DNA
stages of
in the predominantly
(6) that there is a continual
and stimulated
breaks
RESEARCH
repair
levels of NAD’
level of breaks after stimulation
strand
limiting
mice, but the cells did not form
elsewhere
in resting
cellular
not
mitogen
shown
BIOPHYSICAL
structure.
increased
cytes, the latter
factors
ribose polymerase
breaks in the DNA Although
possible
AND
in the steady state
that this is at least partly
and that this causes repair
for
lymphocyte
activation
due
of DNA
and prolifer-
ation.
Acknowledgements This
research
Engineering Greer
Research
has
been
Council
is a post-doctoral
supported
and The
fellow
by
Medical
funded
grants
from
Research
by the Alberta
The
Natural
Science
and
Council
of Canada.
Dr.
Wenda
Heritage
Foundation
for
Medical
Research.
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