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Photoenzymatic repair in mammalian cells revealed by an acridine marker
Vol. 33, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CELLS REVEALED BY AN ACRIDINE MARKER.
PHOTOENZYMATIC REPAIR IN NAMMALIAN Daniel Department
Roth
of Pathology,
560 Firlrt
New
Ave.,
University
York,
N.Y.
Uorpital 10016
Received October 15, 1968
SIBMARY: The UV-dirmerization of thymine and itr photoenaymatic repair were demonstrated in actively growing mamlian cell8 ae well a.9 in inactive deaquamated human buccal cells by an acridine marker rpecific for thymine sequences of intracellular DNA. The etoichiometry of the reaction enabled quantitative measurements of the dye (acriflavine) taken up by the tested cells. The response followed the known kinetics of photoreactivating enzyme derived from bakers’ yeart. Active ryntheris of DNA does not appear to be necessary for this form of photo repair. INTRODUCTION: Photoenzymatic By using of
an acridine
the phenomenon
inactive
repair
marker
cell8
shed
The procedure standardized
single
proposed strand
that
is widely their
changes
as a marker in dye-binding
photodimeritation
limiting under
model,
specificity of
as the major
of acriflavine
conditions
allowing
thir
being
we explored
the potential
for
reaction.
treatment
and photoenzymatic
664
repair
conferred
of UV irradiation thyminer,
cauring
of the dye-binding
clearcut, calculated in
on a
(2i
effect
( 4,5,6),
DNA. (2)
thyminer
molecules.
We found
in a the selective
adjacent
photobiological
paralleled
evidence as in
intracellular
interaction
thymine
cells.(l)
mucora.
at adjacent
which
as well
lines,
similarly,
this
found
amounts
between
for
in mammalian
we have
of the dye with
the adjacent
to be localized,
dirnerization
procedure
both
held
from human buccal
the dye localizes
groups
Inasmuch
photodimer
and two human cell
association
of DNA,
by the S-methyl
thymine
uses
experimental
and near-stoichiometric We have
for
in a murine
squamous
has not been observed
the cellr
replicable to induce we rtudied.
8lOCHEMlCAL
Vol. 33, No. 4, 1968
MATERIALS
AND MM’RODS: Cultured
obtained
from Dr.
University
minimum
essential
Green,
medium
maintained
were
taken
abrasion
by gentle
buffered
under
of 50,000
then washed
conditions
involved
dark
at
supernatant
per
from
for
found
were
were
saline
cell
cleansing
cells
rinse
transferred
were with with
by swirling
into
by hemacytometer
in detail. cells
solution.
The cells
could
UV irradiation
to dryness, (2)
then
was carried
diluted
out
as the reaction
the amount
by measuring
in the
and the unreacted
Inasmuch
and specific,
be determined
this
of freshly
centrifuged
fluorometrically.
count. and stained
Briefly,
to O.OSS)1g Staining
were
was carried 4
“aged”
wett
out
radiation
dose of 7.5 X lo5
were
suspended
by frequent,
quarts
cuvets
in a light-proof
GE G4T4/1
estimated
within
in 0.05M
the
loss
of dye of dye
Results were recorded in picogram8 of dye per cell. f of 57. was obtained with this procedure.
A reproducibility
solution,
10%
of the cell
The buccal
drained
to be near-stoichiometric
the supernate.
kept
with
by suspending
obtained
described
of the unfixed
10 min.
average
an adequately
the WI-38
mucosa of non-smokers
centrifuged,
dye was determined
had been bound
twice,
in buffered 35’C
serum;
and portions
pH 6.0-6.2.
cells
and WI-38 in Eagle’s
supplemented
a preliminary
cells
previously
the exposure
acriflavine
after
cells,
solutfon.
Aliquots They were
medium
New York
maintained
10% calf
of the buccal
The harvested
saline
Biology,
determinations
solution
applicators
were
was performed
dye-binding
obtained
solution.
of Cell
with
basal
passage for
of 3T3 mouse fibroblnsts
HeLa S3 human polyploid
in Eagle’s
saline
saline
consisted
Department
supplemented
phosphate-buffered
cotton-tipped
lines
The 3T3 and HeLa cells
eerum.Atoonfluency,
harvest
cell
of Medicine,
cells.
were
calf
Roward
School
human diploid
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
germicidal ergs/m2.
gentle having
agitation an inside
665
chamber
with
lamp delivering During
irradiation
in 1 ml of buffered diameter
of 1 cm.
an the cells saline
VoL33,No.
BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS
4, 1968
Photoreactivation above
the cells,
its
wae carried light
paering
photoreactivating
enzyme,
at a rtandardiaed
concen,tration
kinetics,
treatment
and 30 min. enzyme After
treutment,
solution
the cell8
and were
then
out
rtained
routinely been
werhed in
glass
b2 (Sigma),
Aeide
of the cells three
the urual
from
at 35'
timer
lamp eet 30 cm
1 cm thick.
wae added
established
requirement8 were
a GE Photoflood
plate
of 314 unite.
dose had
to cover the nurximal
with
through
cytochrome
war carried
The 314 unit
out
to the cell the etudiee
C for
earlier
suepenrion of enzyme
periods
of 10,
to provide
under with
Concurrently,
20,
sufficient
invertigation.
buffered
raline
menner.
TABLE I EFFECTS OF W LIGBT AND PHOTOENZYMTIC REPAIR ON SEVERALCELL TYPES. -
DYE UPTAKE (picogram per cell) CELLTYPE
BASAL
W-IRRADIATED
PHOTOREXTIVATIONAFl!ER UV 10 min 20 min 30 min
~I-38 3s3 BlJXAL-a BIJXAL-b HeLa S3
0.18 0.19
0.12 0.12
0.15 0.18
0.18 0.18
0.18 0.19
0.22 0.24
0.15 0.15
0.20 0.22
0.22 0.24
0.22 0.24
9.35
0.22
0.23
0.23
0.25
TABLE II COMBINATIONS OFWLIGET,
VISlBLELIGHp,ANDENzyME POPUIATIONS.
lX2BUCCALCELL
DYE UPTAKE (picogram per cell) BASAL 0.24 0.22
W-IRRADIATED 0.19
0.15
W plus VISIBLE ENZYMEplus LIGHT (no enzyme) VISIBLE Lt. (no w Lt) 0.19 0.23 0.15 0.22
666
'UV plus VISIBLE Lt &ENZYME 0.24
0.22
BIOCHEMICAL
Vol. 33, No. 4, 1968
AND BlOI+HYSlCAL RESEARCH COMMUNICATIONS
RESULTS :
treated with
Table
I llcltr
cells
in
visible
populations
their
reprerent
LO.
baaal
cell8
the reaction the binding
---- Basal
are is
1):
after the
in picogram8
UV irradiation, three
listed
time
in Table
enzyme-dependent
_
--
_ ”
_
per cell
intervals.
Controls
the
treatment for
2
II.
cells
_
for
and after
fe demonstrated
of dye by buccal
20
Fig.
values
state,
and enzyme over
of buccal That
These
light
the dye-binding
_
in relation
_
-
fn figs.
1 and 2.
to temperature
-
6
40 tEMPER+tURE
60 CC)
Plot of dye uptahe (in picogram per average cell) against photoreactivating temperature. Conridarable enayme activity at S’C; pcah activity at 25’~350; no activity recorded at 65*. Treatment wfth 3/4 Sfm unit8 of yeaat-derived enzyme for 30 min, enryme pre-heated to reaction temperature for 5 min.
667
Vol. 33, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-0e23
-0.22
-0.21
z4 E z.o*lQ
I--
-0,lQ - 0.10 0.10
-
t
-0.17
full CO$CENtRATIC/N
/ Dye uptake with 314 Sigma unlta.
Pig. 2):
dilution
of enzyme,
I rtartlng
and to enzyme concentration,
,and ahow the characteristics
enzyme for
(7)
there
functions.
at full
strength,
of the yeart-derived
DI!JCUSSION: All
tested
cells
except
Rela
exhibited
668
complete
recovery
of
BIOCHEMICAL
Vol. 33, No. 4, 1968
their
basal
dye-binding
enzyme and visible
Prolonging result
in nearly for Ii
it
after
adsorbed of cellular
however,
did
time-rate
thymine
growing
in a number as dark
formation buccal
cells,
it
mamlian
In company
cells
by ultraviolet
and photoenzymatic
cells
wirn ceLr
and repair are well
light.
quantitative
exhibited
is suggested
of mazznalian repair
situated
and photoreactivation
dimer
desquamated
indeed
UNA, these
(a)
caused
the case
significantly.
dye is
and Haynes.
by such mechanisms
injury
with in
a reduced
of DNA replication,
normal
treatment
to 60 minutes,
independent
of UV injury
that
rate
UV-irradiation
for
as exponentially
of Hanawalt
appears
genetic
molecuies
as inactive,
is
recovery
indicating
of the
as a marker
mechanism
of repair human,
their time
part
in dye-binding
Inasmuch
of
the enzyme concentration
recovery,
thymine
to serve
20 minutes
line.
tne greater
same response
concept
not alter
cell
adjacent
“repair”.
repair
Doubling
complete
this
alterations appear
did
after
the photoreactivation
response
between
levels
light.
of the HeLa celLs
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
thus
supporting
other,
recent
lines,
this the reports
including
replication
equipped
the
of MA,
to repair
(9,10,11,12,13)
ACKNOWLEDGMENT: Supported of
the New York
City
by the Division Department
of Cancer
Control
and Research
of Health. REFERENCES :
:; 3) 4) 65; ii,’ 9) 10) 10
12) 13)
Cleaver, J. E. , Biochem. Biophys. Rea. Roth, D., Manjon, ML., and London M “‘z:i: ,‘til%. I’~8”~;ess. Roth, D., and Manjon, M.L., submit;ed;‘(1968). Beukers, R., and Berends, W., Biochim. Biophys. Acta, 41, 550, (1960). Setlow, R.B. , and Setlow, J.K., Nat. Acad. Sci. (U.S.) Proc., 48, 1250, (1962). SetlOW, R.B., Swenson, P.A., and Carrier, W.L., Science, 142, 1464, (1963). Rupert, C.S., J. Gen. Phyaiol., 45, 703, (1962). Hanawalt, P.C., and Haynes, R.H., Biochem. Biophys. Res. COITIIRI~. , 19, 462, (1965). Raszullsen, R.E., and Painter, R.B., J. Cell Biol., 29, 11, (1966). Rauth, A.M., Radiat. Res., 31, 121, (1967). Regan, J.‘D. , and Trosko, J.E., Radiat. Res. 31, 548, (1967). Horikawa, M., Nikaido, O., and Sugahara, T., Nature, 218, 489, (1968). Cleaver, J. E., Nature, 218. 652, (1968).
669
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CELLS REVEALED BY AN ACRIDINE MARKER.
PHOTOENZYMATIC REPAIR IN NAMMALIAN Daniel Department
Roth
of Pathology,
560 Firlrt
New
Ave.,
University
York,
N.Y.
Uorpital 10016
Received October 15, 1968
SIBMARY: The UV-dirmerization of thymine and itr photoenaymatic repair were demonstrated in actively growing mamlian cell8 ae well a.9 in inactive deaquamated human buccal cells by an acridine marker rpecific for thymine sequences of intracellular DNA. The etoichiometry of the reaction enabled quantitative measurements of the dye (acriflavine) taken up by the tested cells. The response followed the known kinetics of photoreactivating enzyme derived from bakers’ yeart. Active ryntheris of DNA does not appear to be necessary for this form of photo repair. INTRODUCTION: Photoenzymatic By using of
an acridine
the phenomenon
inactive
repair
marker
cell8
shed
The procedure standardized
single
proposed strand
that
is widely their
changes
as a marker in dye-binding
photodimeritation
limiting under
model,
specificity of
as the major
of acriflavine
conditions
allowing
thir
being
we explored
the potential
for
reaction.
treatment
and photoenzymatic
664
repair
conferred
of UV irradiation thyminer,
cauring
of the dye-binding
clearcut, calculated in
on a
(2i
effect
( 4,5,6),
DNA. (2)
thyminer
molecules.
We found
in a the selective
adjacent
photobiological
paralleled
evidence as in
intracellular
interaction
thymine
cells.(l)
mucora.
at adjacent
which
as well
lines,
similarly,
this
found
amounts
between
for
in mammalian
we have
of the dye with
the adjacent
to be localized,
dirnerization
procedure
both
held
from human buccal
the dye localizes
groups
Inasmuch
photodimer
and two human cell
association
of DNA,
by the S-methyl
thymine
uses
experimental
and near-stoichiometric We have
for
in a murine
squamous
has not been observed
the cellr
replicable to induce we rtudied.
8lOCHEMlCAL
Vol. 33, No. 4, 1968
MATERIALS
AND MM’RODS: Cultured
obtained
from Dr.
University
minimum
essential
Green,
medium
maintained
were
taken
abrasion
by gentle
buffered
under
of 50,000
then washed
conditions
involved
dark
at
supernatant
per
from
for
found
were
were
saline
cell
cleansing
cells
rinse
transferred
were with with
by swirling
into
by hemacytometer
in detail. cells
solution.
The cells
could
UV irradiation
to dryness, (2)
then
was carried
diluted
out
as the reaction
the amount
by measuring
in the
and the unreacted
Inasmuch
and specific,
be determined
this
of freshly
centrifuged
fluorometrically.
count. and stained
Briefly,
to O.OSS)1g Staining
were
was carried 4
“aged”
wett
out
radiation
dose of 7.5 X lo5
were
suspended
by frequent,
quarts
cuvets
in a light-proof
GE G4T4/1
estimated
within
in 0.05M
the
loss
of dye of dye
Results were recorded in picogram8 of dye per cell. f of 57. was obtained with this procedure.
A reproducibility
solution,
10%
of the cell
The buccal
drained
to be near-stoichiometric
the supernate.
kept
with
by suspending
obtained
described
of the unfixed
10 min.
average
an adequately
the WI-38
mucosa of non-smokers
centrifuged,
dye was determined
had been bound
twice,
in buffered 35’C
serum;
and portions
pH 6.0-6.2.
cells
and WI-38 in Eagle’s
supplemented
a preliminary
cells
previously
the exposure
acriflavine
after
cells,
solutfon.
Aliquots They were
medium
New York
maintained
10% calf
of the buccal
The harvested
saline
Biology,
determinations
solution
applicators
were
was performed
dye-binding
obtained
solution.
of Cell
with
basal
passage for
of 3T3 mouse fibroblnsts
HeLa S3 human polyploid
in Eagle’s
saline
saline
consisted
Department
supplemented
phosphate-buffered
cotton-tipped
lines
The 3T3 and HeLa cells
eerum.Atoonfluency,
harvest
cell
of Medicine,
cells.
were
calf
Roward
School
human diploid
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
germicidal ergs/m2.
gentle having
agitation an inside
665
chamber
with
lamp delivering During
irradiation
in 1 ml of buffered diameter
of 1 cm.
an the cells saline
VoL33,No.
BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS
4, 1968
Photoreactivation above
the cells,
its
wae carried light
paering
photoreactivating
enzyme,
at a rtandardiaed
concen,tration
kinetics,
treatment
and 30 min. enzyme After
treutment,
solution
the cell8
and were
then
out
rtained
routinely been
werhed in
glass
b2 (Sigma),
Aeide
of the cells three
the urual
from
at 35'
timer
lamp eet 30 cm
1 cm thick.
wae added
established
requirement8 were
a GE Photoflood
plate
of 314 unite.
dose had
to cover the nurximal
with
through
cytochrome
war carried
The 314 unit
out
to the cell the etudiee
C for
earlier
suepenrion of enzyme
periods
of 10,
to provide
under with
Concurrently,
20,
sufficient
invertigation.
buffered
raline
menner.
TABLE I EFFECTS OF W LIGBT AND PHOTOENZYMTIC REPAIR ON SEVERALCELL TYPES. -
DYE UPTAKE (picogram per cell) CELLTYPE
BASAL
W-IRRADIATED
PHOTOREXTIVATIONAFl!ER UV 10 min 20 min 30 min
~I-38 3s3 BlJXAL-a BIJXAL-b HeLa S3
0.18 0.19
0.12 0.12
0.15 0.18
0.18 0.18
0.18 0.19
0.22 0.24
0.15 0.15
0.20 0.22
0.22 0.24
0.22 0.24
9.35
0.22
0.23
0.23
0.25
TABLE II COMBINATIONS OFWLIGET,
VISlBLELIGHp,ANDENzyME POPUIATIONS.
lX2BUCCALCELL
DYE UPTAKE (picogram per cell) BASAL 0.24 0.22
W-IRRADIATED 0.19
0.15
W plus VISIBLE ENZYMEplus LIGHT (no enzyme) VISIBLE Lt. (no w Lt) 0.19 0.23 0.15 0.22
666
'UV plus VISIBLE Lt &ENZYME 0.24
0.22
BIOCHEMICAL
Vol. 33, No. 4, 1968
AND BlOI+HYSlCAL RESEARCH COMMUNICATIONS
RESULTS :
treated with
Table
I llcltr
cells
in
visible
populations
their
reprerent
LO.
baaal
cell8
the reaction the binding
---- Basal
are is
1):
after the
in picogram8
UV irradiation, three
listed
time
in Table
enzyme-dependent
_
--
_ ”
_
per cell
intervals.
Controls
the
treatment for
2
II.
cells
_
for
and after
fe demonstrated
of dye by buccal
20
Fig.
values
state,
and enzyme over
of buccal That
These
light
the dye-binding
_
in relation
_
-
fn figs.
1 and 2.
to temperature
-
6
40 tEMPER+tURE
60 CC)
Plot of dye uptahe (in picogram per average cell) against photoreactivating temperature. Conridarable enayme activity at S’C; pcah activity at 25’~350; no activity recorded at 65*. Treatment wfth 3/4 Sfm unit8 of yeaat-derived enzyme for 30 min, enryme pre-heated to reaction temperature for 5 min.
667
Vol. 33, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-0e23
-0.22
-0.21
z4 E z.o*lQ
I--
-0,lQ - 0.10 0.10
-
t
-0.17
full CO$CENtRATIC/N
/ Dye uptake with 314 Sigma unlta.
Pig. 2):
dilution
of enzyme,
I rtartlng
and to enzyme concentration,
,and ahow the characteristics
enzyme for
(7)
there
functions.
at full
strength,
of the yeart-derived
DI!JCUSSION: All
tested
cells
except
Rela
exhibited
668
complete
recovery
of
BIOCHEMICAL
Vol. 33, No. 4, 1968
their
basal
dye-binding
enzyme and visible
Prolonging result
in nearly for Ii
it
after
adsorbed of cellular
however,
did
time-rate
thymine
growing
in a number as dark
formation buccal
cells,
it
mamlian
In company
cells
by ultraviolet
and photoenzymatic
cells
wirn ceLr
and repair are well
light.
quantitative
exhibited
is suggested
of mazznalian repair
situated
and photoreactivation
dimer
desquamated
indeed
UNA, these
(a)
caused
the case
significantly.
dye is
and Haynes.
by such mechanisms
injury
with in
a reduced
of DNA replication,
normal
treatment
to 60 minutes,
independent
of UV injury
that
rate
UV-irradiation
for
as exponentially
of Hanawalt
appears
genetic
molecuies
as inactive,
is
recovery
indicating
of the
as a marker
mechanism
of repair human,
their time
part
in dye-binding
Inasmuch
of
the enzyme concentration
recovery,
thymine
to serve
20 minutes
line.
tne greater
same response
concept
not alter
cell
adjacent
“repair”.
repair
Doubling
complete
this
alterations appear
did
after
the photoreactivation
response
between
levels
light.
of the HeLa celLs
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
thus
supporting
other,
recent
lines,
this the reports
including
replication
equipped
the
of MA,
to repair
(9,10,11,12,13)
ACKNOWLEDGMENT: Supported of
the New York
City
by the Division Department
of Cancer
Control
and Research
of Health. REFERENCES :
:; 3) 4) 65; ii,’ 9) 10) 10
12) 13)
Cleaver, J. E. , Biochem. Biophys. Rea. Roth, D., Manjon, ML., and London M “‘z:i: ,‘til%. I’~8”~;ess. Roth, D., and Manjon, M.L., submit;ed;‘(1968). Beukers, R., and Berends, W., Biochim. Biophys. Acta, 41, 550, (1960). Setlow, R.B. , and Setlow, J.K., Nat. Acad. Sci. (U.S.) Proc., 48, 1250, (1962). SetlOW, R.B., Swenson, P.A., and Carrier, W.L., Science, 142, 1464, (1963). Rupert, C.S., J. Gen. Phyaiol., 45, 703, (1962). Hanawalt, P.C., and Haynes, R.H., Biochem. Biophys. Res. COITIIRI~. , 19, 462, (1965). Raszullsen, R.E., and Painter, R.B., J. Cell Biol., 29, 11, (1966). Rauth, A.M., Radiat. Res., 31, 121, (1967). Regan, J.‘D. , and Trosko, J.E., Radiat. Res. 31, 548, (1967). Horikawa, M., Nikaido, O., and Sugahara, T., Nature, 218, 489, (1968). Cleaver, J. E., Nature, 218. 652, (1968).
669